Modified bio-related substance, process for producing the same, and intermediate

ABSTRACT

A modified bio-related substance, wherein at least one poly(alkylene glycol)oxy group represented by the following formula (1) is combined in a molecule: 
     
       
         
         
             
             
         
       
     
     wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA 1  and OA 2  are each an oxyalkylene group having 2 to 4 carbon atoms, the groups represented by R are the same or different from each other in one molecule, and the groups represented by OA 2  are the same or different from each other in one molecule, n and m are each average number of moles of the oxyalkylene group added, n represents 0 to 1000, and m represents 10 to 1000.

This is Divisional application of application Ser. No. 12/399,249 filedMar. 6, 2009, which is a Continuation application of application Ser.No. 11/142,255 filed Jun. 2, 2005, (now U.S. Pat. No. 7,524,875) whichis a Continuation-In-Part application of U.S. application Ser. No.10/716,432 filed Nov. 20, 2003 (now abandoned); the entire contents ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a bio-related substance modified by thebonding to a polyalkylene glycol derivative, a process for producing thesame, and a reactive polyalkylene glycol derivative which is anintermediate thereof.

BACKGROUND ART

Recently, a large number of proteins, polypeptides, synthetic compounds,and compounds extracted from natural resources having physiologicalactivity and the application thereof to pharmaceuticals has beenextensively studied. However, these physiologically active substanceshave short half-lives in blood when they are injected to a body andhence it is difficult to obtain a sufficient pharmacological effect.This is because the physiologically active substances injected to a bodyare usually cleared from the body because of the filtration throughglomeruli in the kidney and the uptake by macrophages in the liver andspleen. Therefore, it is attempted to improve the behavior in a body byincluding these physiologically active substances in liposomes orpolymer micelles or increasing their molecular weight through chemicalmodification with polyethylene glycol which is an amphiphatic polymer.Polyethylene glycol exhibits a low interaction with the otherbio-components owing to its steric repulsion effect and as a result,proteins and polypeptides such as enzymes modified with polyethyleneglycol exhibit an effect of avoiding the filtration through glomeruli inthe kidney and bio-reactions such as immunoreaction, so that theyachieve half-lives in blood longer than those of unmodified substances.Moreover, they also have decreased toxicity and antigenicity and furtherexhibit an effect of enhancing the solubility of a sparinglywater-soluble compound having a high hydrophobicity.

Hitherto, in the case of modifying a physiologically active substancewith polyethylene glycol, particularly in the case of modifying alow-molecular-weight drug or peptide, there arises a problem that fewreactive functional groups can be used for the modification withpolyethylene glycol. Furthermore, when a peptide or drug is modifiedwith many polyethylene glycol molecules for obtaining a sufficienteffect of the modification with polyethylene glycol, the active site ofthe peptide or drug is blocked and hence problems may arise that its ownfunction and efficacy cannot be exhibited sufficiently and enoughsolubility in water cannot be obtained.

For solving such problems, the reduction of the number of modificationwith polyethylene glycol using a branched polyethylene glycol derivativehas been attempted. JP-B-61-42558 proposes a polyethyleneglycol-modified L-asparaginase. However, cyanuric chloride as a startingmaterial for a reactive polyethylene glycol derivative has threereactive sites and hence it is difficult to introduce two polyethyleneglycol chains thereinto selectively. Accordingly, it is difficult tosynthesize a highly pure polyethylene glycol-modified L-asparaginase.

Also, JP-A-10-67800 proposes a polyethylene glycol-modified interferonα. However, this substance has three urethane and amide bonds includingthe linkage between interferon α and the poly(ethylene glycol) oxygroup. These bonds are labile to hydrolysis during storage or during thereaction under an alkaline condition and as a result, there arises aproblem that the branched polyethylene glycol moiety is decomposed to asingle chain. This is because the polyethylene glycol derivative whichis the intermediate material has been produced by a method wherein twomonomethoxypolyethylene glycols and amino groups at α- and ε-positionsof lysine are combined through urethane bonds and then the carboxylresidue of lysine is converted into a succinimide ester. In addition, inorder to produce the polyethylene glycol-modified interferon α, therearises a problem that increased impurities are produced owing to themulti-step process, such as the activation of the terminal hydroxylgroups of two monomethoxypolyethylene glycols, the combination withlysine, the activation of the carboxyl residue of lysine, and thecombination with interferon α.

Accordingly, it is desired to develop a bio-related substance formed byhighly stable bonds, a process for producing the same, and a branchedreactive polyalkylene glycol derivative which can be produced in aconvenient manner and in a high purity and has a higher stability.

DISCLOSURE OF THE INVENTION

A first object of the invention is to provide a bio-related substancehaving a branched poly(alkylene glycol) oxy group which is formed bystable bonds and is hardly decomposed to a single chain, and a processfor producing the same.

A second object of the invention is to provide a polyalkylene glycolderivative having a reactive group, which can be combined with abio-related substance, at the primary carbon at the 1-position of theglycerin skeleton and having polyalkylene glycol chains at the 2- and3-positions.

As a result of extensive studies for solving the above problems, thepresent inventors have found out a novel bio-related substance having abranched poly(alkylene glycol)oxy group, a process for producing thesame, and a polyalkylene glycol derivative as an intermediate thereof,and thus accomplished the invention.

Namely, the invention relates to a modified bio-related substance,wherein at least one poly(alkylene glycol)oxy group represented by thefollowing formula (1):

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA¹ andOA² are each an oxyalkylene group having 2 to 4 carbon atoms, the groupsrepresented by R are the same or different from each other in onemolecule, and the groups represented by OA² are the same or differentfrom each other in one molecule, n and mare each average number of molesof the oxyalkylene group added, n represents 0 to 1000, and m represents10 to 1000, is combined in a molecule.

Moreover, the invention relates to an intermediate for the modifiedbio-related substance, which is represented by the following formula(2):

wherein R, OA¹, OA², n, and m are the same as above, and X represents afunctional group capable of chemically reacting with a bio-relatedsubstance.

Furthermore, the invention relates to a process for producing a modifiedbio-related substance wherein at least one poly(alkylene glycol) oxygroup represented by the formula (1) is combined in a molecule,

comprising a step of combining the above intermediate with a bio-relatedsubstance.

In addition, the invention relates to a compound of the formula (p) as astarting material of the compound of the formula (2) and a process forproducing the same.

The modified bio-related substance of the invention is formed by stablebonds and is hardly decomposed to a single chain. Moreover, theinvention can provide a polyalkylene glycol derivative having a reactivegroup, which can be combined with a bio-related substance, at theprimary carbon at the 1-position of the glycerin skeleton and havingpolyalkylene glycol chains at the 2- and 3-positions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an experimental result by polyacrylamide gelelectrophoresis of OVA and modified OVA.

FIG. 2 is a chart illustrating a result of GPC measurement before anaccelerated aging test of the compound p-8.

FIG. 3 is a chart illustrating a result of GPC measurement after anaccelerated aging test of the compound p-8.

FIG. 4 is a chart illustrating a result of GPC measurement before anaccelerated aging test of the compound p-10.

FIG. 5 is a chart illustrating a result of GPC measurement after anaccelerated aging test of the compound p-10.

FIG. 6 is a result of electrophoresis of the compound obtained bymodifying Humanin with the compound (p31).

FIG. 7 is a result of electrophoresis of the compound obtained bymodifying insulin with the compound (p32) or (p35).

BEST MODE FOR CARRYING OUT THE INVENTION

The modified bio-related substance of the invention is a substancewherein a bio-related substance is combined with at least onepoly(alkylene glycol)oxy group represented by the above formula (1).

R in the poly(alkylene glycol)oxy group of the formula (1) is ahydrocarbon group having 1 to 24 carbon atoms and specific hydrocarbongroups include hydrocarbon groups such as a methyl group, an ethylgroup, a propyl group, an isopropyl group, a butyl group, a tert-butylgroup, a pentyl group, an isopentyl group, a hexyl group, a heptylgroup, a 2-ethylhexyl group, an octyl group, a nonyl group, a decylgroup, an undecyl group, a dodecyl group, a tridecyl group, a tetradecylgroup, a pentadecyl group, a hexadecyl group, a heptadecyl group, anoctadecyl group, an oleyl group, a nonadecyl group, an eicosyl group, aheneicosyl group, docosyl group, a tricosyl group, a tetracosyl group, abenzyl group, a cresyl group, a butylphenyl group, and a dodecylphenylgroup. The hydrocarbon group is preferably a hydrocarbon group having 1to 10 carbon atoms, more preferably a methyl group or an ethyl group,further preferably a methyl group.

OA¹ and OA² represent each an oxyalkylene group having 2 to 4 carbonatoms. Specifically, they include an oxyethylene group, an oxypropylenegroup, an oxytrimethylene group, an oxy-1-ethylethylene group, anoxy-1,2-dimethylethylene group, and an oxytetramethylene group. Theoxyalkylene groups may be the same or different from each other and maybe added randomly or block-wise. In general, the fewer the carbon atomsare, the higher the hydrophilicity is. The group is preferably anoxyethylene group or an oxypropylene group, more preferably anoxyethylene group m and n are each average number of moles of theoxyalkylene group added. m represents 10 to 1000, preferably 20 to 800,more preferably 50 to 800, most preferably 100 to 800. n represents 0 to1000, preferably 0 to 500, more preferably 0 to 200, most preferably 0to 50. In a preferable embodiment, n is 0. In another preferableembodiment, n is 1 to 50. In the latter case, n is particularlypreferably 1 to 3.

The number of modifications with the poly(alkylene glycol)oxy group tothe bio-related substance is not particularly limited but is preferably1 to 100, more preferably 1 to 20.

The “bio-related substance” according to the invention means a substancerelating to a body. The substances relating to a body include thefollowing.

(1) Animal Cell-Constituting Materials Such as Phospholipids,Glycolipides, and Glycoproteins

The animal cell-constituting materials are components constituting cellmembranes and the kind is not particularly limited but examples thereofinclude phospholipids, glycolipides, and glycoproteins. Examples of morespecific phospholipids include phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, cardiolipin, phosphatidylserine, andphosphatidylinositol. In addition, lyso isomers thereof are alsoincluded. These phospholipids may be those derived from natural productssuch as egg yolk or soybean or may be synthesized products. Thecomposition of fatty acids is not particularly limited but may includefatty acids having 12 to 22 carbon atoms. These fatty acids may besaturated fatty acids or may be those containing an unsaturated bond.Examples of more specific glycolipids include ceramides, cerebrosides,sphingosines, gangliosides, and glyceroglycolipids. In addition, fattyacids, monoglycerides, diglycerides, cholesterols, and bile acid arealso included.

(2) Body Fluid-Constituting Substances Such as Blood, Lymph, and BoneMarrow Liquid

The body fluid-constituting substances mean fluid components existinginside and outside cells and the kind is not particularly limited butexamples thereof include blood, lymph, and bone marrow liquid. Examplesof more specific body fluid-constituting components include hemoglobin,albumin, and blood coagulation factors.

(3) Physiologically Active Substances Such as Vitamins,Neurotransmitters, Proteins, Polypeptides, and Drugs

The physiologically active substances mean components controlling bodyfunctions and the kind is not particularly limited but examples thereofinclude vitamins, neurotransmitters, proteins, polypeptides, and drugs.

Examples of more specific vitamins include vitamin A, vitamin B, vitaminC, vitamin D, vitamin E, and vitamin K.

Examples of more specific neurotransmitters include adrenalin,noradrenalin, dopamine, acetylcholine, GABA, glutamic acid, and asparticacid.

Examples of more specific proteins and polypeptides include thefollowing. Hormones such as neurohypophysial hormone, thyroid hormone,male sex hormone, female sex hormone, and adrenal cortex hormone. Serumproteins such as hemoglobin and blood factors Immunoglobulins such asIgG, IgE, IgM, IgA, and IgD. Cytokines and fragments thereof, such asinterleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-S, IL-9,IL-10, IL-11 and IL-12 subtypes), interferons (−α, −β, −γ),granulocyte-colony stimulating factors (α and β types),macrophage-colony stimulating factor, granulocyte-macrophage colonystimulating factor, platelet-derived growth factor,phospholipase-activating protein, insulin, glucagon, lectin, ricin,tumor necrosis factor, epidermal growth factor, transforming growthfactors (−α, −β), fibroblast growth factor, hepatocyte growth factor,vascular endothelial growth factor, nerve growth factor, bone growthfactor, insulin-like growth factor, heparin binding growth factor, tumorgrowth factor, glial cell line-derived neurotrophic factor, macrophagedifferentiating factor, differentiation-inducing factor, leukemiainhibitory factor, amphiregurin, somatomedin, erythropoietin,hemopoietin, thrombopoietin, and calcitonin. Enzymes such as proteolyticenzymes, oxidoreductases, transferases, hydrases, lyases, isomerases,ligases, asparaginases, arginases, arginine deaminases, adenosinedeaminases, superoxide dismutases, endotoxinases, catalases,chymotrypsin, lipases, uricases, elastases, streptokinases, urokinases,prourokinases, adenosine diphosphatases, tyrosinases, bilirubinoxidases, glucose oxidases, glucodases, glactosidases,glucocerebrosidases, and glucouronidases. Monoclonal and polyclonalantibodies and fragments thereof. Polyamino acids such as poly-L-lysine,poly-D-lysine. Vaccines such as hepatitis B vaccine, malaria vaccine,melanoma vaccine, and HIV-1 vaccine, and antigens. In addition,glycoproteins are also included. Furthermore, also included arestructurally similar substances having physiological activity similar tothat of these physiologically active substances.

Moreover, these proteins and polypeptides may be isolated from naturalsources thereof or cells subjected to genetic engineering or may beproduced via various synthetic processes.

The drugs are not particularly limited but more preferably includeanticancer drugs and antifungal drugs.

More specific anticancer drugs are not particularly limited but, forexample, include paclitaxel, adriamycin, doxorubicin, cisplatin,daunomycin, mitomycin, vincristine, epirubicin, methotrexate,5-fluorouracil, aclacinomycin, idamycin, bleomycin, pirarubicin,peplomycin, vancomycin, and camptothecine.

Specific antifungal drugs are not particularly limited but, for example,include amphotericin B, nystatin, flucytosine, miconazole, fluconazole,itraconazole, ketoconazole, and peptide antifungal drugs.

Moreover, these physiologically active substances also includeflavonoids, terpenoids, carotenoids, saponins, steroids, quinones,anthraquinones, xanthones, coumarins, alkaloids, porphyrins, andpolyphenols.

The intermediate for the bio-related substance of the invention isrepresented by the following formula (2).

In the formula, X is not particularly limited as far as it is afunctional group or an unsaturated bond capable of forming a chemicalbond with a bio-related substance. In a preferable embodiment, X is agroup represented by the group (I), (II) or (III).

In the case of the reaction with an amino group of a bio-relatedsubstance, the groups represented by (a), (b), (d), (f), (h), (i), and(k) are preferable. In the case of the reaction with a mercapto group ofa bio-related substance, the groups represented by (a), (b), (c), (d),(e), (f), (h), (i), (k), and (xx1) are preferable. In the case of thereaction with an unsaturated bond of a bio-related substance, the grouprepresented by (c) is preferable. In the case of the reaction with acarboxyl group of a bio-related substance, the groups represented by(c), (g), and (j) are preferable. Moreover, in the case of the reactionwith the aldehyde group of the bio-related substance, the groupsrepresented by (c), (g), (j), (xx2), and (xx3) are preferable. In thecase that the bio-related substance does not have any of an amino group,a mercapto group, an unsaturated bond, a carboxyl group, and an aldehydegroup, these groups may be suitably introduced therein.

Z in the group (I), (II) or (III) is a linker between the poly(alkyleneglycol) oxy group and the reactive functional group and is notparticularly limited as far as it is a covalent bond, but preferablyincludes an alkylene group and an alkylene group containing an esterbond, a urethane bond, an amide bond, an ether bond, a carbonate bond,or a secondary amino group. Preferable alkylene group includes amethylene group, an ethylene group, a trimethylene group, a propylenegroup, an isopropylene group, a tetramethylene group, a butylene group,an isobutylene group, a pentamethylene group, and a hexamethylene group.More preferable is a structure of the following (z1). More preferable asan alkylene group containing an ester bond is a structure of thefollowing (z2).

More preferable as an alkylene group containing an amide bond is astructure of the following (z3). A group of the following (z4), (Z7) and(z8) are more preferable as an alkylene group containing an ether bond.More preferable as an alkylene group containing a urethane bond is astructure of the following (z5). The following (z6) is a structure morepreferable as an alkylene group containing a secondary amino group. s isdefined for each formula independently, and s is an integer of 1 to 6,preferably an integer of 1 to 3, more preferably an integer of 2 to 3.

Y is a hydrocarbon group having 1 to 10 carbon atoms which may containfluorine atom(s). Specifically, Y includes a methyl group, an ethylgroup, a propyl group, an isopropyl group, a butyl group, a tert-butylgroup, a hexyl group, a nonyl group, a vinyl group, a phenyl group, abenzyl group, a 4-methylphenyl group, a trifluoromethyl group, a2,2,2-trifluoroethyl group, and a 4-(trifluoromethoxy)phenyl group, andis preferably a methyl group, a vinyl group, a 4-methylphenyl group, anda 2,2,2-trifluoroethyl group.

W¹ in the group (III) is a halogen atom selected from Cl, Br and I, andis preferably I.

In the compound represented by the formula (2), R, A¹O, A²O, n, and mare the same as above.

Z, R, A¹O, A²O, n, and m in the group (II) and the group (III) are alsothe same as above.

Tables 1, 2 and 6 show relation between a residual group T of the abovebio-related substance and a functional group X of the poly(alkyleneglycol)oxy group side which forms a chemical bond with the residualgroup T. In addition, Tables 1, 2 and 6 also show types of the chemicalbonds between the poly(alkylene glycol)oxy groups and bio-relatedsubstances, which are formed by the reaction of the bio-relatedsubstances with X.

TABLE 1 Reactive group of physiologically active substance X group NH₂—TAmino group SH—T Mercapto group

(OA¹)—S—T (sulfide)

—Z—SH (c)

—Z—S—S—T (disulfide)

Note: In (b) and (d), X group is a moiety excluding (OA¹).

TABLE 2 Reactive group of physiologically active substance X group NH₂—TAmino group SH—T Mercapto group

—Z—NH₂ (g), (j)

—Z—COOH (k)

Note: In (h) and (i), X group is a moiety excluding (OA¹).

TABLE 6 Reactive group of physiologically active substance X group NH₂—TAmino group SH—T Mercapto group

—Z—ONH₂ (xx3)

As is apparent from the tables, in the modified bio-related substancesof the invention, the poly(alkylene glycol)oxy group and the bio-relatedsubstance are combined by, for example, an amide bond, a secondary aminogroup, a urethane bond, a thioester bond, a sulfide bond, a disulfidebond, a thiocarbonate bond, an oxime, a hydrazone bond, or a thioacetalbond.

The modified bio-related substances of the invention can be produced asfollows.

(Case of Reacting an Amino Group of a Bio-Related Substance with anIntermediate of the Invention)

In the case of the modification with an amino group of a bio-relatedsubstance, the intermediates (a), (b), (d), (f), (h), (i), and (k) ofthe invention are used. More preferably, (a), (b), (d), and (f) areused. At the reaction, the intermediates (a), (b), (d), (f), (h), (i),and (k) of the invention may be reacted in a ratio of equimolar or moreto the bio-related substance. The reaction solvent is not particularlylimited as far as it does not participate in the reaction, but in thecase of reacting a protein or polypeptide, preferable solvents includebuffer solutions such as phosphate buffer solutions, borate buffersolutions, Tris-acid buffer solutions, acetate buffer solutions, andcarbonate buffer solutions. Furthermore, an organic solvent which doesnot deactivate the protein or polypeptide and does not participate inthe reaction, such as acetonitrile, dimethyl sulfoxide,dimethylformamide, or dimethylacetamide, may be added. In the case ofreacting an anticancer drug, antifungal drug, or phospholipid,preferable solvents include, in addition to the above buffer solutions,toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether,t-butyl methyl ether, tetrahydrofuran, chloroform, methylene dichloride,dimethyl sulfoxide, dimethylformamide, dimethylacetamide, water,methanol, ethanol, n-propanol, 2-propanol, and n-butanol. Also, thesolvent need not be used. The order of adding the intermediate and thebio-related substance is optional. The reaction temperature is notparticularly limited as far as it does not deactivate the bio-relatedsubstance, but the temperature is preferably 0 to 40° C. in the case ofreacting a protein or polypeptide and is preferably −20 to 150° C. inthe case of reacting an anticancer drug, antifungal drug, orphospholipid. The reaction time is preferably 0.5 to 72 hours, morepreferably 1 to 24 hours. At the reaction, a condensing agent such asN,N′-dicyclohexylcarbodiimide (DCC) or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) may beused. A covalent bond is formed between the bio-related substance andthe intermediate of the invention by carrying out the reaction. An amidebond is formed in the case of using (a) or (k), a secondary amino groupin the case of using (b), a urethane bond in the case of using (d), (h),or (i), and a Schiff base in the case of using (f). When a Schiff baseis formed, it may be subjected to a reduction treatment using a reducingagent such as sodium cyanoborohydride to form a secondary amino group.After the reaction, the product may be purified by a purifying meanssuch as dialysis, salting-out, ultrafiltration, ion-exchangechromatography, electrophoresis, extraction, recrystallization,adsorption treatment, reprecipitation, column chromatography, orsupercritical extraction.

(Case of Reacting a Mercapto Group of a Bio-Related Substance with anIntermediate of the Invention)

In the case of the modification with a mercapto group of a bio-relatedsubstance, the intermediates (a), (b), (c), (d), (e), (f), (h), (i),(k), and (xx1) of the invention are used. More preferably, (e) and (xx1)are used. The reaction solvent, reaction conditions, and the like arethe same as in the case of using an amino group. At the reaction, aradical initiator such as iodine or AIBN may be used. A covalent bond isformed between the bio-related substance and the intermediate of theinvention by carrying out the reaction, and a thioether bond is formedin the case of using (a) or (k), a thiocarbonate bond in the case ofusing (d), (h), or (i), a disulfide bond in the case of using (c), and asulfide bond in the case of using (b), (e), (f), or (xx1).

(Case of Reacting an Unsaturated Bond of a Bio-Related Substance with anIntermediate of the Invention)

In the case of the modification with an unsaturated bond of abio-related substance, the intermediate (c) of the invention is used.The reaction solvent, reaction conditions, and the like are the same asin the case of using an amino group. At the reaction, a radicalinitiator such as iodine or AIBN may be used. A sulfide bond is formedbetween the bio-related substance and the intermediate of the inventionby carrying out the reaction.

(Case of Reacting a Carboxyl Group of a Bio-Related Substance with anIntermediate of the Invention)

In the case of the modification with a carboxyl group of a bio-relatedsubstance, the intermediate (a), (g), or (j) of the invention is used.The reaction solvent, reaction conditions, and the like are the same asin the case of using an amino group. At the reaction, a condensing agentsuch as DCC or EDC may be optionally used. A covalent bond is formedbetween the bio-related substance and the intermediate of the inventionby carrying out the reaction, and a thioester bond is formed in the caseof using (c) and an amide bond in the case of using (g) or (j).

(Case of Reacting an Aldehyde Group of a Bio-Related Substance with anIntermediate of the Invention)

In the case of the modification with an aldehyde group of a bio-relatedsubstance, the intermediates (c), (g), (j), (xx2), and (xx3) of theinvention are used. The reaction solvent, the reaction conditions, andthe like are the same as in the case of using the amino group. When aSchiff base is formed, it may be subjected to a reduction treatmentusing a reducing agent such as sodium cyanoborohydride. By carrying outthe reaction, a thioacetal bond is formed in the case of using (c), asecondary amino group in the case of using (g) or (j), an oxime in thecase of using (xx2), and a hydrozone bond in the case of using (xx3).

Moreover, in the case that a bio-related substance does not have any ofan amino group, a mercapto group, an unsaturated bond, a carboxyl group,and an aldehyde group, the substance can be modified by introducing areactive group suitably into the bio-related substance and using anintermediate of the invention.

(Production of Intermediates)

The intermediates of the invention can be, for example, produced asfollows. An alkylene oxide is polymerized in an amount of 0 to 1000 molto the primary hydroxyl group residue of2,2-dimethyl-1,3-dioxolane-4-methanol and the terminal hydroxyl group isprotected with a benzyl group or a t-Bu group. Thereafter, the cyclicacetal structure is deprotected under an acidic condition and analkylene oxide is polymerized in an amount of 10 to 1000 mol to thenewly formed two hydroxyl groups, followed by alkyl-etherification ofthe terminal ends. Then, the protective group such as the benzyl groupor t-Bu group is deprotected and thereby, the compound of the generalformula (p) can be obtained. When n is 0, the primary hydroxyl groupresidue of 2,2-dimethyl-1,3-dioxolane-4-methanol is protected with abenzyl group or t-Bu group and then an alkylene oxide is polymerized inan amount of 10 to 1.000 mol to the newly formed two hydroxyl groups,followed by alkyl-etherification of the terminal ends. Then, theprotective group such as the benzyl group or t-Bu group is deprotectedand thereby, the compound of the general formula (p) can be obtained.

Alternatively, the compound (p) can be also produced by the followingmethod. The primary hydroxyl group of2,2-dimethyl-1,3-dioxolane-4-methanol is protected by a benzyl group ort-Bu group. Thereafter, the cyclic acetal structure is deprotected underan acidic condition and an alkylene oxide is polymerized in an amount of10 to 1000 mol to the newly formed two hydroxyl groups, followed byalkyl-etherification of the terminal ends. Then, the protective groupsuch as the benzyl group or t-Bu group is deprotected and thereby, thecompound of the general formula (p) wherein n is 0 can be obtained. Thecompound may be also produced by polymerizing an alkylene oxide in anamount of 0 to 1000 mol to the newly formed hydroxyl group.

When n is 1 to 3, after coupling of2,2-dimethyl-1,3-dioxolane-4-methanol with 2-benzyloxyethanol (n=1),diethylene glycol benzyl ether (n=2), or triethylene glycol benzyl ether(n=3), the cyclic acetal structure is deprotected under an acidiccondition and an alkylene oxide is polymerized in an amount of 10 to1000 mol to the newly formed two hydroxyl groups, followed byalkyl-etherification of the terminal ends. Then, the protective groupsuch as the benzyl group or t-Bu group is deprotected and thereby, thecompound of the general formula (p) can be obtained.

As above, a highly pure branched polyalkylene glycol derivative can beproduced in high yields in an industrially suitable manner by using thealkylene oxide-addition polymerization reaction, without columnpurification.

Using the hydroxyl group of the compound (p) thus obtained, theintermediates of the invention can be produced by modifying hydroxygroup into various reactive groups shown in the groups (I), (II) and(III). Furthermore, using the formed reactive groups, variousbio-related substances can be reacted and modified to produce modifiedbio-related substances of the invention.

Moreover, the intermediate having each functional group of the groups(I), (II) and (III) can be reacted with a bio-related substance but insome cases, the intermediate can be further reacted with the othercompound to produce other intermediate and the other intermediate can bethen reacted with a bio-related substance. For example, using theintermediate having a functional group (g), (j), or (k) belonging to thegroup (II) as an starting material, the intermediate having (a), (e), or(f) of the group (I) can be synthesized.

The addition polymerization of an alkylene oxide to the primary hydroxylgroup residue of 2,2-dimethyl-1,3-dioxolane-4-methanol can be carriedout in the following manner. The addition polymerization of anoxyalkylene can be achieved in toluene or without solvent under analkaline condition such as sodium, potassium, sodium hydride, potassiumhydride, sodium methoxide, or potassium t-butoxide. In the case that nis 1. to 3, the step of the addition polymerization of alkylene oxideneed not be carried out. The subsequent benzyl etherification can becarried out in the following manner.

1) It can be achieved by reacting benzyl chloride or benzyl bromide with2,2-diraethyl-1,3-dioxolane-4-methanol or its alkylene oxide adduct inan aprotic solvent or without any solvent in the presence of an alkalicatalyst such as sodium hydroxide or potassium hydroxide.2) It can be achieved by converting the hydroxyl group of2,2-dimethyl-1,3-dioxolane-4-methanol or its alkylene oxide adduct in anaprotic solvent or without any solvent using sodium, potassium, sodiumhydride, potassium hydride, sodium methoxide, potassium methoxide,potassium t-butoxide, or the like into an alcoholate and reacting thealcoholate with benzyl chloride or benzyl bromide under a basiccondition.3) It can be achieved by activating the hydroxyl group of2,2-dimethyl-1,3-dioxolane-4-methanol or its alkylene oxide adduct withmethanesulfonyl chloride, p-toluenesulfonyl chloride,2,2,2-trifluoroethanesulfonyl chloride, or the like in an aproticsolvent or without any solvent, followed by the reaction with analcoholate of benzyl alcohol.4) It can be achieved by activating the hydroxyl group of2-benzyloxyethanol (n=1), diethylene glycol benzyl ether (n=2), ortriethylene glycol benzyl ether (n=3) with methanesulfonyl chloride,p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride, orthe like in an aprotic solvent or without any solvent, followed by thereaction with an alcoholate of 2,2-dimethyl-1,3-dioxolane-4-methanol.

The deprotection of the cyclic acetal structure which follows the benzyletherification is achieved by the reaction in an aqueous solutionadjusted to pH 1 to 4 with an acid such as acetic acid, phosphoric acid,sulfuric acid, or hydrochloric acid, whereby a compound of the formula(9) can be produced.

The addition polymerization of an alkylene oxide to the compound of thefollowing formula (9) having two hydroxyl groups newly formed by thedeprotection of the cyclic acetal is not particularly limited but can beachieved via the following steps (C1) and (C2).

Step (C1): The alcoholation of the compound of the formula (9) isachieved by using sodium or potassium, preferably sodium as a catalystin an catalyst amount of 5 to 50 moles, followed by dissolution at 10 to50° C.

Step (C2): An alkylene oxide addition polymerization is carried out at areaction temperature of 50 to 130° C.

With regard to the catalyst amount in the step (C1), since thepolymerization rate of the alkylene oxide decreases at less than 5 molesand heat history increases to result in the formation of impurities suchas a terminal vinyl ether compound, the use of the catalyst in an amountof 5 mol % or more is advantageous in the production of a high qualityhigh-molecular-weight compound. When the catalyst amount exceeds 50 mol%, the viscosity of the reaction liquid increases or the liquidsolidifies at the alcoholation reaction and thus there is a tendencythat the stirring efficiency decreases and the alcoholation is notaccelerated. Moreover, when the liquid solidifies, handling thereoftends to be difficult, which causes water absorption. When thealcoholate has absorbed water, an alkylene glycol compound derived fromwater is formed and is contained as an impurity undesirable in medicaluse.

When the temperature at the dissolution is higher than 50° C., adecomposition reaction may occur to form benzyl alcohol and glycerin.When benzyl alcohol is formed, it initiates addition polymerization withthe alkylene oxide, whereby a low-molecular-weight impurity having amolecular weight 0.5 time the molecular weight of the target compound.When the low-molecular-weight impurity derived from benzyl alcohol isformed, a functional group is introduced via alkyl-etherification of thehydroxyl group and deprotection in the subsequent steps as in the caseof the target compound, so that the impurity is converted into alow-molecular-weight impurity which is reactive with a bio-relatedsubstance. There is the possibility that such impurity may react with abio-related substance and change the physical properties of theresulting preparation. Moreover, when glycerin is formed, it alsoinitiates addition polymerization with the alkylene oxide to form ahigh-molecular-weight impurity having a molecular weight 1.5 times thatof the target compound. Since the high-molecular-weight impurity doesnot have a benzyl group and its terminal hydroxyl group is onlyalkyl-etherified, no functional group is introduced. However, when thecombination with a drug or the like is carried out while such impurityis contained, the resulting preparation becomes inhomogeneous and hencethe quality tends to be varied. Also, the preparation is not suitable ina medical use where a highly pure product is required.

When the dissolution is carried out at a temperature lower than 10° C.,like the case that the catalyst amount is more than 50 mol %, theviscosity of the reaction liquid increases or the liquid solidified atthe alcoholation reaction, and handling thereof tends to be difficult,and water absorption is caused.

The reaction solvent is not particularly limited as far as it is anaprotic solvent such as toluene, benzene, xylene, acetonitrile, ethylacetate, tetrahydrofuran, chloroform, methylene dichloride, dimethylsulfoxide, dimethylformamide, or dimethylacetamide, but preferable istoluene or no solvent. The reaction time is preferably 1 to 24 hours.When the time is less than 1 hour, there is the possibility that thecatalyst does not completely dissolved. When the time is longer than 24hours, there is the possibility that the above decomposition reactionmay occur.

With regard to the reaction temperature in the step (C2), when thetemperature is lower than 50° C., the polymerization rate is low andheat history increases to result in a tendency to decrease the qualityof the compound of the formula (5). Moreover, when the temperature ishigher than 130° C., side reactions such as vinyl etherification of theterminal end occur during the polymerization and thus the quality of thetarget compound tends to decrease. During the polymerization, as themolecular weight increases, the viscosity of the reaction liquid alsoincreases, so that an aprotic solvent, preferably toluene may beoptionally added.

As another production process in the step of alcoholation, the followingstep (C3) may be mentioned.

Step (C3): Sodium methoxide, potassium t-butoxide, or potassiummethoxide, preferably sodium methoxide is added as an catalyst in anamount of 5 to 50 mol % and the reaction is carried out at 60 to 80° C.At that time, a pressure-reducing operation may be conducted in order tofacilitate the exchange reaction.

The catalyst amount is preferably 5 to 50 mol % for the reason mentionedabove. With regard to the reaction temperature, when the temperature islower than 60° C., the conversion of the exchange reaction decreases andalcohols such as methanol remain, which leads to the formation ofimpurities having a molecular weight 0.5 time that of the targetcompound. When the temperature is higher than 80° C., a degradationreaction occurs. The alcoholation reaction requires elevation of thetemperature and the reaction time is desirably 1 to 3 hours since thedegradation reaction is apt to occur. When the time is shorter than 1hour, there is the possibility that the conversion into the alcoholatedecreases. When the time is longer than 3 hours, the decomposition mayoccur. The reaction solvent is not particularly limited as far as it isan aprotic solvent, but preferable is toluene or no solvent.

The subsequent alkyl-etherification of the terminal end may be achievedby either of the following (1) or (2):

(1) a process of converting the terminal end of the polyalkylene glycolchain into an alcoholate and reacting it with an alkyl halide;(2) a process of activating the terminal hydroxyl group of thepolyalkylene glycol chain with methanesulfonyl chloride,p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride, orthe like, followed by the reaction with an alcoholate of alkyl alcohol.

Preferable is the process (2) and the following will describe it in moredetail.

The production process (2) comprises the following steps (B1), (B2), and(B3).Step (B1): A step of adding a dehalogenating agent and a compoundrepresented by the formula (6) to a compound represented by the formula(5) and reacting them at 20 to 60° C. to obtain a compound of theformula (7). At that time, each charged molar ratio satisfies thefollowing relationship:

Vc≧3Va

Vb>vc

Va: number of moles of the compound represented by the formula (5)Vb: number of moles of the dehalogenating agentVc: number of moles of the compound represented by the formula (6).

More preferable is the case that each charged molar ratio satisfies thefollowing relationship:

20Va≧Vc≧3Va

4Vc>Vb>Vc.

When Vc is smaller than 3Va, the conversion decreases and thus some partof the hydroxyl groups in the oxyalkylene chain terminal ends remainunchanged. A functional group is introduced to the remaining hydroxylgroup to form a polyfunctional impurity having a molecular weight thesame as that of the target compound. When such polyfunctional impurityis present, it acts as a crosslinking agent at the combination with abio-related substance to result in a tendency to decrease the purity ofthe resulting modified bio-related substance. When Vb is not larger thanVc, the conversion decreases owing to inefficient trapping of an acidwhich is produced as a by-product with the progress of the reaction, sothat some part of the hydroxyl groups in the oxyalkylene chain terminalends remain unchanged. Moreover, when Vc is larger than 20Va or Vb isnot smaller than 4Vc, an excess of each reagent or compound may becontained to cause side reactions in the subsequent processes.

The dehalogenating agent to be used includes organic bases such astriethylamine, pyridine, and 4-dimethylaminopyridine, and inorganicbases such as sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium carbonate, and potassium hydroxide.Preferable dehydrochlorinating agent is an organic base such astriethylamine, pyridine, or 4-dimethylaminopyridine.

In the compound of the formula (6) to be used, W is preferably Cl or Br,and R¹ is preferably a methyl group, a phenyl group, or a p-methylphenylgroup. More suitably, methanesulfonyl chloride where W is Cl and R¹ is amethyl group is most preferable.

The solvent to be used at that time is not particularly limited as faras it is an aprotic solvent and preferably includes toluene, benzene,xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform,methylene dichloride, dimethyl sulfoxide, dimethylformamide, ordimethylacetamide, but more preferable is toluene which enablesazeotropic removal of water in the system. The amount of the solvent tobe used at the reaction is preferably 0.5 equivalent weight to 10equivalent weight to the compound of the formula (5). In the case thatthe compound of the formula (5) has a large molecular weight, theviscosity of the reaction liquid increases and the conversion decreases,so that it is preferable to dilute the reaction liquid with the solvent.

The reaction temperature is not particularly limited but is preferably60° C. or lower for the purpose of inhibiting side reactions and ispreferably 20° C. or higher for the purpose of inhibiting increase ofthe viscosity of the reaction liquid. The reaction time is preferably 1to 24 hours. When the time is less than 1 hour, there is the possibilitythat the conversion is low. When the time is longer than 24 hours, thereis the possibility that a side reaction may occur.

At the reaction, the operation of removing water from the startingmaterials, such as azeotropic removal of water may be carried out priorto the reaction. Moreover, an antioxidant such as2,6-di-tert-butyl-p-cresol may be added. Furthermore, a salt is formedwith the progress of the reaction and the formation of the compound ofthe formula (7), but the reaction mixture may be used in the subsequentstep as it is, or the salt may be removed by filtration, or after thefiltration, the compound of the formula (7) may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

Step (B2): A step of adding a compound represented by the formula (8) tothe compound of the formula (7) and reacting them at 20 to 80° C. toobtain the compound of the formula (4). At that time, each charged molarratio satisfies the following relationship:

Vd>Vc

Vd: number of moles of the compound represented by the formula (8).

More preferable is the case that the relationship:

10Vc>Vd>Vc

is satisfied.

R—OM  (8)

In the formula (8), R is as mentioned above and M is sodium orpotassium, preferably sodium.

When Vd is not larger than Vc, the alkyl-etherification does notsufficiently proceed and a reactive group such as a mesylate groupremains unchanged at the oxyalkylene chain terminal end. When a reactivegroup remains at the oxyalkylene chain terminal end, as mentioned above,a polyfunctional compound is formed and a serious side reaction iscaused at the combination with a bio-related substance. Moreover, whenVd is not smaller than 10Vc, an excess of the alcoholate may becontained to cause side reactions in the subsequent process.

The solvent to be used in the reaction is not particularly limited asfar as it is an aprotic solvent and is preferably toluene. The amount ofthe solvent to be used at the reaction is preferably an amount of 0.5equivalent to 10 equivalent to the compound of the formula (7). In thecase that the compound of the formula (7) has a large molecular weight,the viscosity of the reaction liquid increases, so that it is preferableto dilute the reaction liquid with the solvent.

The reaction temperature is not particularly limited but is preferably80° C. or lower for the purpose of inhibiting side reactions and ispreferably 20° C. or higher for the purpose of inhibiting increase ofthe viscosity of the reaction liquid. The reaction time is preferably 1to 24 hours. When the time is less than 1 hour, there is the possibilitythat the conversion is low. When the time is longer than 24 hours, thereis the possibility that a side reaction occurs. At the reaction, anoperation of removing water from the starting materials, such asazeotropic removal of water may be carried out prior to the reaction.

Step (B3): A step of filtrating the reaction liquid or washing thereaction liquid with an aqueous inorganic salt solution having aconcentration of 10 wt % or more.

In the step, the inorganic salt is not particularly limited but ispreferably sodium chloride. When the concentration is less than 10 wt %,the target compound migrates into an aqueous layer to decrease theprocess yield remarkably. The operation of washing with water may berepeated several times. The step (B3) is carried out for removingstarting materials excessively added and salts produced as by-products.The omission of the step may cause side reactions in the case that thesteps (B1) to (B3) are again carried out in the next place. In the casethat a debenzylation step is carried out as a next step, theseimpurities act as catalyst poisons and thus the conversion may beaffected.

Moreover, in order to enhance the ratio of alkyl-etherification of theoxyalkylene chain terminal end, it is preferable to repeat the steps(B1) to (B3) again. When the ratio of alkyl-etherification of theoxyalkylene chain terminal end is low, as mentioned above, there is thepossibility of forming a polyfunctional impurity.

The compound of the formula (4) thus obtained may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

The production of the compound (p) by successive debenzylation is notparticularly limited but it can be produced by hydrogenation of thefollowing step (A) using a hydrogenative reduction catalyst and ahydrogen donor.

Step (A): A step of subjecting the compound represented by the formula(4) to a hydrogenative reduction reaction under the condition that thewater content in the reaction system is 1% or less. When the watercontent in the reaction system is more than 1%, the decompositionreaction of the polyoxyalkylene chain occurs. Since polyalkylene glycolformed by the decomposition has a hydroxyl group, it is functionalizedin the next step to form a reactive low-molecular-weight impurity. Suchreactive low-molecular-weight impurity reacts with a bio-relatedsubstance as mentioned above and thus tends to change the properties ofthe resulting preparation.

The hydrogenative reduction catalyst is preferably palladium. Thesupport is not particularly limited but is preferably alumina or carbon,more preferably carbon. The amount of palladium is preferably 1 to 20 wt% based on the compound of the formula (4). When the amount is less than1 wt %, the conversion of deprotection decreases and thus there is thepossibility that the ratio of functionalization, in the next stepdecreases. Moreover, when the amount is more than 20 wt %, thedecomposition reaction of the polyalkylene glycol chain may occur andthere is the possibility that the above reactive low-molecular-weightcompound is produced as a by-product. The reaction solvent is notparticularly limited as far as the water content in the reaction systemis less than 1%, but preferably includes methanol, ethanol, 2-propanol,and the like and more preferable is methanol. The hydrogen donor is notparticularly limited but include hydrogen gas, cyclohexene, 2-propanol,and the like. The reaction temperature is preferably 40° C. or lower.When the temperature is higher than 40° C., the decomposition reactionof the polyalkylene glycol chain may occur and there is the possibilitythat the reactive low-molecular-weight compound is produced as aby-product. The reaction time is not particularly limited. When largeamount of the catalyst is used, the reaction is completed within a shortperiod of time. But, when the amount is small, a longer period of timeis required. In general, the reaction time is preferably 1 to 5 hours.When the time is shorter than 1 hour, there is the possibility that theconversion is low. When it is longer than 5 hours, the decompositionreaction of the poly(alkylene glycol) may occur.

The resulting compound of the formula (p) may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

The thus obtained compound is a polyalkylene glycol derivativerepresented by the following formula (p) and containing substantially nosecondary hydroxyl group:

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA¹ andOA² are each an oxyalkylene group having 2 to 4 carbon atoms, the groupsrepresented by R are the same or different from each other in onemolecule, and the groups represented by OA² are the same or differentfrom each other in one molecule, n and m are each average number ofmoles of the oxyalkylene group added, n represents 0 to 1000, and mrepresents 10 to 1000.

Since the compound of the formula (p) contains no secondary hydroxylgroup, the conversion of the subsequent functional group-introducingreaction is high and a highly pure polyalkylene glycol derivative can beobtained. In the case that a secondary hydroxyl group is present, theconversion of the subsequent functional group-introducing reaction islow and the purity of intermediate of the modified bio-related substancedecreases, so that there may arise the problem of contamination of thedrug or the like with an impurity.

The compound of the formula (p) of the invention satisfies therelationship:

Hrd/Mp×1000000≦3

wherein Mp is a molecular weight corresponding to the peak top obtainedfrom gel-permeation chromatography of the polyalkylene glycol derivativeof the formula (p), and Hrd is a ratio of remaining hydroxyl groupcontained in the alkyl group R at the polyoxyalkylene chain terminal endin the 2- and 3-positions.

More preferably, it satisfies the relationship:

Hrd/Mp×1000000≦2.

Mp means a weight-average molecular weight at the point of the maximumrefractive index among peaks excluding the peaks caused by a developingsolvent used in gel-permeation chromatography and false peaks derivedfrom base line fluctuation caused by the column and apparatus used. Inthe invention, gel permeation chromatography is carried out using SHODEXGPC SYSTEM-11 as a GPC system and measurement was conducted under thefollowing conditions:

developing solvent: tetrahydrofuran; flow rate: 1 ml/min; column: SHODEXKF-801, KF-803, KF-804 (I.D. 8 mm×30 cm); column temperature: 40° C.;detector: RI×8; sample amount: 1 mg/g, 100 μl.

The ratio Hrd of the remaining hydroxyl group contained in the alkylgroup R is measured after mesylation of the compound of the formula (4)which is a precursor before deprotection. The following will illustratethe case that R is a methyl group.

5Ve g of toluene is added to Ve g of the compound of the formula (4),followed by removal of water azeotropically under normal pressure. Aftercooling to 40° C., 20 mol of triethylamine is added to 1 mol of thecompound of the formula (4) and after thorough stirring, 6 mol ofmethanesulfonyl chloride is added thereto. At that time, it is desirableto add it dropwise after dilution with toluene or without dilution.Then, the reaction is carried out at 40° C. for 3 hours andtriethylamine salt of methanesulfonic acid is removed by filtration.Then, 10Ve to 20Ve g of ethyl acetate is added to the filtrate and aftercooling to room temperature, hexane is gradually added until crystalsprecipitate. The resulting crystals are collected by filtration and 10Veto 20Ve g of ethyl acetate is again added to the crystals, followed byheating to dissolve them. After cooling to room temperature, hexane isgradually added until crystals precipitate. The crystals are collectedby filtration and dried. A 20 mg portion of the resulting dried productis dissolved in deuterated chloroform and ¹H nuclear magnetic resonancespectrum is measured. Hrd is represented by the following relationship:

Hrd=Mms/(Mms+Mme)

wherein Mme is an integral value of peak of the methyl group of theoxyalkylene chain terminal end detected at 3.38 ppm and Mms is anintegral value of peak of the mesyl group formed by mesylating theremaining hydroxyl group of the oxyalkylene chain terminal end, which isdetected at 3.08 ppm, a TMS base peak being 0 ppm.

When R is a group other than a methyl group, Hrd can be determined bysuitably identifying a peak position where the alkyl group is detectedand applying a similar equation in consideration of the proton number.

When Hrd thus determined satisfies the following relationship:

Hrd/Mp×1000000>3,

the case means that a large amount of impurities where hydroxyl groupsremain at 2- and 3-positions of the polyoxyalkylene chain terminal endare contained. When such impurities are present, the hydroxyl group ofthe polyoxyalkylene chain terminal end are also functionalized in thesubsequent step to form polyfunctional impurities. Such impurities mayact as crosslinking agents at the combination with a bio-relatedsubstance as mentioned above to cause side reactions.

With regard to the compound of the formula (p) of the invention,polydispersity Mw/Mn in all the peaks from the starting point of elutionto the end point of elution satisfies the relationship:

Mw/Mn≦1.07

at the measurement of gel permeation chromatography. More preferable isthe case that the relationship:

Mw/Mn≦1.05

is satisfied.

In the case that Mw/Mn is larger than 1.07, it means the presence of alarge amount of the above-mentioned high-molecular-weight impurities andlow-molecular weight impurity and when the compound is combined with abio-related substance, there is the possibility that the formation of byproducts increases to result in an insufficient purity. Moreover, whenthe purity is insufficient, the product may cause an adverse effect whenused as an medical product.

The compound of the formula (p) of the invention satisfies therelationships

M2/(M1+M2)×100≦10

wherein M1 is an integral value of the methyl group detected at around3.13 ppm, which is originated from the mesyl group derived from thehydroxyl group at the 1-position directly bonded to the glycerinskeleton in the case that n is 0 when the compound is reacted withmethanesulfonyl chloride to obtain a mesylated compound and a nuclearmagnetic resonance spectrum thereof is measured as a deuterated methanolsolution, andM2 is an integral value of the methyl group detected at around 3.12 ppm,which is originated from the mesyl group derived from the hydroxyl groupof the polyalkylene glycol chain. More preferably, the relationships

M2/(M1+M2)×100≦8

is satisfied.

The following will illustrate the calculation method of M1 and M2.

4Vf g of toluene is added to Vf g of the compound of the formula (p),followed by removal of water azeotropically under normal pressure. Aftercooling to 40° C., 20 mol of triethylamine is added to 1 mol of thecompound of the formula (p) and after thorough stirring, 6 mol ofmethanesulfonyl chloride is added thereto. At that time, it is desirableto add it dropwise after dilution with toluene or without dilution.Then, the reaction is carried out at 40° C. for 3 hours andtriethylamine salt of methanesulfonyl chloride is removed by filtration.Thereafter, 10Vf to 20Vf g of ethyl acetate is added to the filtrate andafter cooling to room temperature, hexane is gradually added untilcrystals precipitate. The resulting crystals are collected by filtrationand 10Vf to 20Vf g of ethyl acetate is again added to the crystals,followed by heating to dissolve them. After cooling to room temperature,hexane is gradually added until crystals precipitate. The crystals arecollected by filtration and dried. A 20 mg portion of the resultingdried product is dissolved in deuterated methanol and ¹H nuclearmagnetic resonance spectrum is measured. M1 is determined as an integralvalue of the methyl group detected at around 3.13 ppm, which isoriginated from the mesyl group derived from the hydroxyl group at the1-position directly bonded to the glycerin skeleton in the case that nis 0, a TMS base peak being 0 ppm. Moreover, M2 is determined as anintegral value of the methyl group detected at around 3.12 ppm, which isoriginated from the mesyl group derived from the polyalkylene glycolchain terminal end or polyalkylene glycol chain formed by thedecomposition reaction.

In the case that the relationship:

M2/(M1+M2)×100>10

which is derived from M1 and M2 thus determined, is satisfied, thepurity of the resulting modified bio-related substance tends to decreasebecause the substance is contaminated with a large amount of theimpurities shown below.

That is, the case means that a large amount of impurities having ahydroxyl group at the polyoxyalkylene chain terminal end, which areoriginated from the impurities:

(A): an impurity having a hydroxyl group and a molecular weight 0.5 timethat of the compound (p), which is formed by decomposition of thecompound of the formula (9) at the alcoholation, addition polymerizationof an alkylene oxide to the resulting benzyl alcohol, and deprotectionof benzyl group in the subsequent step;(B): an impurity having a remaining hydroxyl group at 2- or 3-positionand a molecular weight the same as that of the compound (p), which isformed at the alkyl-etherification of the compound of the formula (5);(C): an impurity having a hydroxyl group and a low molecular weight,which is formed by decomposition of the polyoxyalkylene chain at thedebenzylation of the compound of the formula (4); and the like, arepresent.

The debenzylation reaction of the invention is widely applicable toother derivatives.

More specifically, it is a process for producing a polyalkylene glycolderivative of the formula (11), comprising the following step (AA):

Step (AA): a step of subjecting a compound represented by the formula(10) to a hydrogenative reduction reaction under the condition that thewater content in the reaction system is 1% or less:

wherein G is a residual group of a compound having 2 to 4 hydroxylgroups; R² is a hydrocarbon group having 1 to 4 carbon atoms; m1, m2,and m3 represent each average number of moles of an oxyethylene groupadded and satisfy the following relationship:

0≦m1≦1000, 0≦m2≦1000, 0≦m3≦1000, 10≦m1+m2+m3≦1000;

x¹ is an amino group, a carboxyl group, or a protected group thereof;and g1, g2, and g3 represent each an integer and satisfy the followingrelational equations:

1≦g1≦3, 0≦g2, 0≦g3, 2≦g1+g2+g3≦4.

More specific residual group of a compound having 2 to 4 hydroxyl groupsin G includes ethylene glycol, glycerin, pentaerythritol, diglycerin,and the like, and more preferable is ethylene glycol or glycerin.

More specific R² includes a methyl group, an ethyl group, a propylgroup, an isopropyl group, a t-butyl group, and the like, and preferableis a methyl group.

With regard to m1, m2, and m3, they are not particularly limited as faras the relationships:

0≦m1≦1000, 0≦m2≦1000, 0≦m3≦1000, 10≦m1+m2+m3≦1000

are satisfied, but preferable is the case of 20≦m1+m2+m3≦1000, morepreferably is the case of 40≦m1+m2+m3≦1000, and most preferable is thecase of 100≦m1+m2+m3≦1000.

Specific x¹ includes an amino group, a Boc amino group, an Fmoc aminogroup, a carboxyl group, and the like, and more preferable is a Bacamino group, wherein Boa means a t-butoxycarbonyl group and Fmoc means a9-fluorenylmethoxycarbonyl group.

The water content in the reaction system, catalyst amount, reactiontime, solvent, and the like are the same as those in the aforementionedstep (A). The hydrogenative reduction reaction can be carried out usinga hydrogenative reduction catalyst. The hydrogenative reduction catalystis preferably palladium.

The alkyl-etherification of the invention is widely applicable to otherderivatives.

More specifically, it is a process for producing a polyalkylene glycolderivative represented by the formula (16), wherein the following steps(BB1) to (BB3) are carried out.

Step (BB1): A step of adding a dehalogenating agent and a compoundrepresented by the formula (14) to a compound represented by the formula(12) and reacting them at 20 to 60° C. to obtain a compound of theformula (13). At that time, each charged molar ratio satisfies thefollowing relationship:

Vj≧1.5×Vh×g5

Vi>Vj

Vh: number of moles of the compound represented by the formula (12)Vi: number of moles of the dehalogenating agentVj: number of moles of the compound represented by the formula (14).

Step (BB2): A step of adding a compound represented by the formula (15)to a compound of the formula (13) and reacting them at 20 to 80° C. toobtain a compound of the formula (16). At that time, each charged molarratio satisfies the following relationship:

Vk>Vj

Vk: number of moles of the compound represented by the formula (15):

wherein G, m1, m2, m3, and x¹ are the same as above, and g4, g5, and g6represent each an integer and satisfy the following relationalequations:

0≦g4, 1≦g5≦3, 0≦g6, 2≦g4+g5+g6≦4.

wherein G, m1, m2, m3, and x¹ are the same as above, W is a halogen atomselected from Cl, Br and I, and R³ is a hydrocarbon group having 1 to 10carbon atoms.

R²—OM  (15)

wherein R² is the same as above and M is potassium or sodium.

wherein G, R², m1, m2, m3, and x² are the same as above.

Step (BB3): A step of filtrating the reaction liquid or washing thereaction liquid with an aqueous inorganic salt solution having aconcentration of 10 wt % or more.

In the compound of the formula (14), W is preferably Cl or Br, and R³ ispreferably a methyl group, a phenyl group, or a p-methylphenyl group,and most preferable is methanesulfonyl chloride where W is C¹ and R³ isa methyl group.

The inorganic salt is not particularly limited but is preferably sodiumchloride.

Moreover, for the aforementioned reasons, in order to enhance the ratioof alkyl-etherification of the oxyethylene chain terminal end, it ispreferable to repeat again the steps (BB1) to (BB3).

The water content in the reaction system, catalyst amount, reactiontime, solvent, and the like are the same as the aforementioned steps(B1) to (B3).

The following shows the reaction pathways to the compound

The following will describe the introduction of a reactive group intothe hydroxyl group of the compound (p) formed by the debenzylationreaction.

(Process for Producing (b), (d), (h), and (i))

By reacting the compound (p) with an organic base such as triethylamine,pyridine, or 4-dimethylaminopyridine or an inorganic base such as sodiumcarbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate,potassium carbonate, or potassium hydroxide and any one of the compoundsrepresented by the following general formulae (b1), (d1), (h1), and (i1)in an aprotic solvent such as toluene, benzene, xylene, acetonitrile,ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran,chloroform, methylene dichloride, dimethyl sulfoxide, dimethylformamide,or dimethylacetamide or without any solvent, (b), (d), (h), and (i) canbe introduced, respectively. Moreover, the above organic base orinorganic base need not be used. The ratio of the organic base orinorganic base to be used is not particularly limited but is preferablyequimolar or more to the compound (p). Furthermore, an organic base maybe used as a solvent. W in (b1) or (d1) is a halogen atom selected fromCl, Br and I, and is preferably Cl. The ratio of the compoundsrepresented by the general formulae (b1), (d1), (h1), and (i1) to beused is not particularly limited but is preferably equimolar or more,more preferably equimolar to 50 molar to the compound (p). The reactiontemperature is preferably 0 to 300° C., more preferably 20 to 150° C.The reaction time is preferably 10 minutes to 48 hours, more preferably30 minutes to 24 hours. The compound formed may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

wherein W is a halogen atom selected from Cl, Br and I.

(Process for Producing (a) and (k))

The succinimide compound (a) can be obtained by reacting the compound(p) with a dicarboxylic acid anhydride such as succinic anhydride orglutaric anhydride to obtain a carboxyl compound (k), followed bycondensation with N-hydroxysuccinimide in the presence of a condensingagent such as DCC or EDC. The reaction of the compound (p) with adicarboxylic acid anhydride is carried out in the aforementioned aproticsolvent or without any solvent. The ratio of the dicarboxylic acidanhydride to be used is not particularly limited but is preferablyequimolar or more, more preferably equimolar to 5 molar to the compound(p). The reaction temperature is preferably 0 to 200° C., morepreferably 20 to 150° C. The reaction time is preferably 10 minutes to48 hours, more preferably 30 minutes to 12 hours. In the reaction, anorganic base such as triethylamine, pyridine, or dimethylaminopyridineor an inorganic base such as sodium carbonate, sodium hydroxide, sodiumhydrogen carbonate, sodium acetate, potassium carbonate, or potassiumhydroxide may be used as a catalyst. The ratio of the catalyst to beused is preferably 0.1 to 50 wt %, more preferably 0.5 to 20 wt %. Thecarboxyl compound (k) thus formed may be purified by the aforementionedpurification means or may be used as it is in the next condensationreaction.

The subsequent condensation reaction is also carried out in theaforementioned aprotic solvent or without any solvent. The condensingagent is not particularly limited but is preferably DCC. The ratio ofDCC to be used is preferably equimolar or more, more preferablyequimolar to 5 molar to the compound (p). The ratio ofN-hydroxysuccinimide to be used is preferably equimolar or more, morepreferably equimolar to 5 molar to the compound (p). The reactiontemperature is preferably 0 to 100° C., more preferably 20 to 80° C. Thereaction time is preferably 10 minutes to 48 hours, more preferably 30minutes to 12 hours. The compound formed may be purified by theaforementioned purification means.

The compound (a) can be obtained also by the flowing method. It can beobtained by reacting the compound (p) with N,N′-disuccinimidylcarbonate. The reaction of the compound (p) with N,N′-disuccinimidylcarbonate is carried out in the aforementioned aprotic solvent orwithout any solvent. The ratio of N,N′-disuccinimidyl carbonate to beused is not particularly limited but is preferably equimolar or more,more preferably equimolar to 20 molar to the compound (p). The reactiontemperature is preferably 0 to 200° C., more preferably 20 to 150° C.The reaction time is preferably 10 minutes to 48 hours, more preferably30 minutes to 12 hours. In the reaction, an organic base such astriethylamine, pyridine, or dimethylaminopyridine or an inorganic basesuch as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate,sodium acetate, potassium carbonate, or potassium hydroxide may be usedas a catalyst. The ratio of the catalyst to be used is preferably 0.1 to50 wt %, more preferably 0.5 to 20 wt %. The compound (a) thus formedmay be purified by the aforementioned purification means.

(Process for Producing (g) and (j))

The amine compound (g) can be obtained by reacting the compound (p) toacrylonitrile or the like using an inorganic base such as sodiumhydroxide or potassium hydroxide in a solvent such as water andacetonitrile to obtain a nitrile compound and then subjecting it tohydrogenation of the nitrile group in the presence of a nickel orpalladium catalyst in an autoclave. The ratio of the inorganic base tobe used for obtaining the nitrile compound is not particularly limitedbut is preferably 0.01 to 50 wt % to the compound (p). The ratio ofacrylonitrile or the like to be used is not particularly limited but ispreferably 0.5 to 5 equivalent weight, more preferably 1 to 4 equivalentweight to the weight of the compound (p). Moreover, acrylonitrile may beused as a solvent. The reaction temperature is preferably −50 to 100°C., more preferably −20 to 60° C. The reaction time is preferably 10minutes to 48 hours, more preferably 30 minutes to 24 hours. Thereaction solvent in the subsequent hydrogenation reaction is notparticularly limited as far as it does not participate in the reaction,but is preferably toluene. The ratio of the nickel or palladium catalystto be used is not particularly limited but is 0.05 to 30 wt %,preferably 0.5 to 20 wt % to the nitrile compound. The reactiontemperature is preferably 20 to 200° C., more preferably 50 to 150° C.The reaction time is preferably 3.0 minutes to 48 hours, more preferably30 minutes to 24 hours. The hydrogen pressure is preferably 2 to 10 MPa,more preferably 3 to 8 MPa. Moreover, in order to prevent dimerization,ammonia may be added to the reaction system. In the case of addingammonia, the ammonia pressure is not particularly limited but is 0.1 to10 MPa, more preferably 0.3 to 2 MPa. The compound formed may bepurified by the aforementioned purification means.

The above amine compound (g) or (j) can be also obtained by reacting (b)with aqueous ammonia. The reaction is carried out in aqueous ammonia andthe concentration of ammonia is not particularly limited but ispreferably in the range of 10 to 40%. The ratio of aqueous ammonia to beused is preferably 1 to 300 times the weight of (b). The reactiontemperature is preferably 0 to 100° C., more preferably 20 to 80° C. Thereaction time is preferably 10 minutes to 72 hours, more preferably 1hour to 36 hours. Alternatively, the amine compound (g) or (j) can bealso obtained by reacting (b) with ammonia in an autoclave. The reactionsolvent is not particularly limited but preferably includes methanol andethanol. The amount of ammonia is preferably 10 to 300 wt %, morepreferably 20 to 200 wt %. The reaction temperature is preferably 50 to200° C., more preferably 80 to 150° C. The reaction time is preferably10 minutes to 24 hours, more preferably 30 minutes to 12 hours. Thecompound formed may be purified by the aforementioned purificationmeans.

(Process for Producing (e))

Furthermore, the maleimide compound (e) can be obtained by reacting theresulting amine (g) with maleic anhydride in the aforementioned aproticsolvent or without any solvent to obtain an maleamide compound and thensubjecting it to a ring closure reaction using acetic anhydride orsodium acetate. The ratio of maleic anhydride to be used in themaleamidation reaction is not particularly limited but is preferablyequimolar or more, more preferably equimolar to 5 molar to the compound(p). The reaction temperature is preferably 0 to 200° C., morepreferably 20 to 120° C. The reaction time is preferably 10 minutes to48 hours, more preferably 30 minutes to 12 hours. The maleamide compoundformed may be purified by the aforementioned purification means or maybe used as it is in the next ring closure reaction.

The reaction solvent in the subsequent ring closure reaction is notparticularly limited but is preferably aprotic solvent or aceticanhydride. The ratio of sodium acetate to be used is not particularlylimited but is preferably equimolar or more, more preferably equimolarto 50 molar to the maleamide compound. The reaction temperature ispreferably 0 to 200° C., more preferably 20 to 150° C. The reaction timeis preferably 10 minutes to 48 hours, more preferably 30 minutes to 12hours. The compound formed may be purified by the aforementionedpurification means.

The above maleimide compound can be also obtained by reacting thecompound of the following formula (e1) with the aforementioned amine (g)or (j). The reaction is carried out in the aforementioned aproticsolvent or without any solvent and the compound (e1) is added in anamount of equimolar or more to the amine (g) or (j). The ratio of thecompound (e1) to be used is preferably equimolar or more, morepreferably equimolar to 5 molar to the amine (g) or (j). The reactiontemperature is preferably 0 to 200° C., more preferably 20 to 80° C. Thereaction time is preferably 10 minutes to 48 hours, more preferably 30minutes to 12 hours. During the reaction, light shielding may beconducted. The compound formed may be purified by the aforementionedpurification means.

wherein Q represents a hydrocarbon group having 1 to 7 carbon atoms.

(Process for Producing (f))

The aldehyde compound (f) can be obtained by reacting the compound (b)with an acetal compound (f1) to obtain an acetal compound and thensubjecting it to hydrolysis under an acidic condition. The compound (b)is produced as mentioned above. The acetalization reaction can beachieved by reacting the compound (b) with an equimolar or more amount,preferably an equimolar to 50 molar amount of the compound (f1) in theaforementioned aprotic solvent or without any solvent. The compound (f1)can be prepared from the corresponding alcohol using sodium, potassium,sodium hydride, sodium methoxide, potassium t-butoxide, or the like. Thereaction temperature is preferably 0 to 300° C., more preferably 20 to150° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to 24 hours.

In the case of using the compound (f2), an acetal compound can beobtained by converting the hydroxyl group of the compound (p) into analcoholate by the aforementioned method and then reacting it with anequimolar or more amount, preferably an equimolar to 100 molar amount ofthe compound (f2) in the aforementioned aprotic solvent or without anysolvent. The reaction temperature is preferably 0 to 300° C., morepreferably 20 to 150° C. The reaction time is preferably 10 minutes to48 hours, more preferably 30 minutes to 24 hours.

In the case of using the compound (f3), an acetal compound can beobtained by reacting the compound (f3) with the compound (a), (b), (d),(h), (i), or (k). The compound (a), (b), (d), (h), (i), or (k) isproduced as mentioned above. In the reaction with the compound (f3), thesolvent is not particularly limited but the reaction is preferablycarried out in the aforementioned aprotic solvent. The charging ratio ofthe compound (f3) is preferably equimolar or more, more preferablyequimolar to 10 molar to the compound (a), (b), (d), (h), (i), or (k).The reaction temperature is preferably −30 to 200° C., more preferably 0to 150° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to 24 hours. In the case of using the compound(k), a condensing agent such as DCC or EDC may be optionally used. Anyacetalization reaction may be carried out under light shielding. Theacetal compound thus obtained may be purified by the aforementionedpurification means or may be used as it is in the nextaldehyde-formation reaction.

The aldehyde compound can be produced by hydrolyzing the acetal compoundin a 0.1 to 50% aqueous solution which is adjusted to pH 1 to 4 with anacid such as acetic acid, phosphoric acid, sulfuric acid, orhydrochloric acid. The reaction temperature is preferably −20 to 100°C., more preferably 0 to 80° C. The reaction time is preferably 10minutes to 24 hours, more preferably 30 minutes to 10 hours. Thereaction may be carried out under light shielding. The compound formedmay be purified by the aforementioned purification means.

wherein R¹ and R² are each a hydrocarbon group having 1 to 3 carbonatoms and may be the same or different from each other, and they maytogether form a ring; M is sodium or potassium; W is a halogen atomselected from Cl, Br, and I; and t is an integer of 1 to 5.

(Process for Producing (c))

The mercapto compound (c) can be obtained by reacting the compound (b)with a thiol-forming agent such as thiourea. The compound (b) isproduced as mentioned above. The thio-formation reaction is carried outin a solvent such as water, an alcohol, or acetonitrile or without anysolvent. The ratio of thiourea to be used is equimolar or more, morepreferably equimolar to 50 molar to the compound (b). The reactiontemperature is preferably 0 to 300° C., more preferably 20 to 150° C.The reaction time is preferably 10 minutes to 48 hours, more preferably30 minutes to 24 hours. After the reaction, the mercapto compound can beobtained by subjecting the resulting thiazolium salt to alkalihydrolysis. The compound formed may be purified by the aforementionedpurification, means.

Moreover, the above mercapto compound can be also obtained by reactingthe compound (b) with the following compound (c1), followed bydecomposition with a primary amine. The reaction of the compound (b)with the compound (c1) is carried out in the aforementioned aproticsolvent or without any solvent. The ratio of the compound (c1) to beused is equimolar or more, more preferably equimolar to 50 molar to thecompound (b). The reaction temperature is preferably 0 to 300° C., morepreferably 20 to 80° C. The reaction time is preferably 10 minutes to 48hours, more preferably 30 minutes to 24 hours. The subsequent alkalidecomposition with a primary amine is carried out in the aforementionedaprotic solvent or without any solvent. The primary amine to be used isnot particularly limited but preferably includes ammonia, methylamine,ethylamine, propylamine, butylamine, pentylamine, hexylamine,cyclohexylamine, ethanolamine, propanolamine, and butanolamine.Naturally, the primary amine may be used as a solvent. The compoundformed may be purified by the aforementioned purification means.

(Production Method of (xx1))

The haloacetyl compound (xx1) can be, for example, produced by thefollowing method. It can be obtained by reacting the compound (p), (g)or (j) with a compound represented by the following general formula(xx1a) and an organic bases such as triethylamine, pyridine, and4-dimethylaminopyridine or an inorganic base such as sodium carbonate,sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassiumcarbonate, or potassium hydroxide in an aprotic solvent such as toluene,benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butylmethyl ether, tetrahydrofuran, chloroform, methylene dichloride,dimethyl sulfoxide, dimethylformamide, or dimethylacetamide or withoutany solvent. The ratio of the organic base or inorganic base to be usedis not particularly limited but is preferably equimolar or more to thecompound (p), (g) or (j). Moreover, an organic base may be used as asolvent. The organic or inorganic base is optionally used. W¹ in (xx1a)is a halogen atom selected from Cl, Br and I, and is preferably I.Furthermore, W¹'s may be the same or different from each other. Theratio of the compound represented by the formula (xx1a) to be used isnot particularly limited but is preferably equimolar or more, morepreferably equimolar to 50 molar to the compound (p), (g) or (j). Thereaction temperature is preferably 0 to 300° C., more preferably 20 to150° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to 24 hours. The compound formed may be purifiedby a purification means such as extraction, recrystallization,adsorption treatment, reprecipitation, column chromatography, orsupercritical extraction.

(Production Method of (xx2))

The compound (xx2) can be, for example, produced by the followingmethod. The hydrazine derivative (xx2) can be obtained by condensing thecompound (k) with the following compound (xx2a) in the presence of acondensing agent such as DCC, EDC, or BOP [(benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate]. The reaction of thecompound (p) with (xx2a) is carried out in the aforementioned aproticsolvent or without any solvent. The ratio of the (xx2a) to be used isnot particularly limited but is preferably equimolar or more, morepreferably equimolar to 5 molar to the compound (p). The condensingagent is not particularly limited but is preferably DCC, EDC, or BOP.The ratio of the condensing agent to be used is preferably equimolar ormore, more preferably equimolar to 5 molar to the compound (p). Thereaction temperature is preferably 0 to 100° C., more preferably 20 to80° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to hours. The compound formed may be purified bythe aforementioned purification means.

NH₂NH-Boc  (xx2a)

wherein Boc represents a t-butoxycarbonyl group.

The subsequent deprotection of the Boc group can be achieved by a knownmethod. The compound (xx2) synthesized may be purified by theaforementioned purification means.

(Production Method of (xx3))

The compound (xx3) can be, for example, produced by the followingmethod. The hydroxylamine derivative (xx3) can be obtained by condensingthe compound (p), (g), or (j) with the following compound (xx3a) in thepresence of a condensing agent such as DCC, EDC, or BOP. The reaction ofthe compound (p) with (xx3a) is carried out in the aforementionedaprotic solvent or without any solvent. The ratio of the (xx3a) to beused is not particularly limited but is preferably equimolar or more,more preferably equimolar to 5 molar to the compound (p). The condensingagent is not particularly limited but is preferably DCC, EDC, or BOP.The ratio of the condensing agent to be used is preferably equimolar ormore, more preferably equimolar to 5 molar to the compound (p). Thereaction temperature is preferably 0 to 100° C., more preferably 20 to80° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to 12 hours. The compound formed may be purifiedby the aforementioned purification means.

The subsequent deprotection of the Boo group can be achieved by a knownmethod. The compound (xx3) synthesized may be purified by theaforementioned purification means.

According to the invention, a bio-related substance modified with abranched poly(alkylene glycol)oxy group can be obtained. The bio-relatedsubstance is formed by ether bonds only except for the linker part withthe poly(alkylene glycol)oxy group, so that a high stability can beexpected with no decomposition to a single chain. Therefore, bymodifying a bio-related substance with a branched polyalkylene glycol, abio-related substance exhibiting an improved behavior in a body can beprovided. The intermediate of the bio-related substance of the inventionis a novel compound having a reactive group, which can be combined witha bio-related substance, at the primary carbon at the 1-position of theglycerin skeleton and having polyalkylene glycol chains at the 2- and3-positions.

EXAMPLES

The following will describe the invention more specifically based onExamples. In this regard, ¹H-NMR and GPC were employed for analyzing andidentifying the compounds in Examples.

Method for ¹H-NMR Analysis:

At ¹H-NMR analysis, JNM-ECP400 manufactured by Nippon Denshi Datum K.K.The integral values in NMR data are theoretical values.

Method for GPC Analysis:

At GPC analysis, SHODEX GPC SYSTEM-11 was employed as a GPC system andmeasurement was carried out under the following conditions:

developing solvent: tetrahydrofuran; flow rate: 1 ml/min; column: SHODEXKF-801, KF-803, RF-804 (I.D. 8 mm×30 cm); column temperature: 40° C.;detector: RIX 8; sample amount: 1 mg/g, 100 μl.

In GPC data, analysis values at main peaks which are obtained by cuttingelution curves perpendicular to base lines at inflection points toremove high-molecular-weight impurities and low-molecular-weightimpurities and analysis values over whole peaks from start points ofelution to end points of elution.

Mn represents a number average molecular weight, Mw a weight averagemolecular weight, and Mp a peak top molecular weight.

For the measurement of water content, a Karl Fisher's moisture meter(788/3-20 type manufactured by Metrome-Shibata) was employed and“HYDRANAL-composite 2” manufactured by Sigma Aldrich was employed as aKarl Fisher's reagent.

Example 1 Synthesis of Compound (p) (Case of R=Methyl Group, A¹O,A²O=Oxyethylene Group, n=0, and Molecular Weight=about 10000) Example1-1

To a 1000 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 132.2 g (1.0 mol) of2,2-dimethyl-1,3-dioxolane-4-methanol, 231.4 g (1.2 mol) of a 28%methanol solution of sodium methoxide, and 500 ml of toluene. Withintroduction of nitrogen thereinto, the toluene was refluxed underreduced pressure for 1 hour to remove the methanol by distillation. Withmaintaining the solution at 80° C., 126.6 g (1.0 mol) of benzyl chloridewas added dropwise over a period of 2 hours using a dropping funnel,followed by further 2 hours of reaction. The solvent was removed fromthe reaction liquid and the residue was purified by distillation (b.p.93-95° C./266 Pa) to obtain4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane.

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 1.36, 1.42 (3H, 3H, s,C(CH₃ )₂), 3.45-3.57 (2H, m, CH₂ O—C(CH₃)₂), 3.73-3.76 (1H, m,CHO—C(CH₃)₂), 4.03-4.07, 4.28-4.32 (2H, m, CH₂ O—CH₂Ph), 4.57 (2H, q,—CH₂ Ph), 7.15-7.40 (5H, m, —CH₂ Ph) (Ph represents a phenyl group)

Example 1-2

Into a 1 L beaker were weighed 222 g (1.0 mol) of4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane purified in Example 1-1,250 ml of ethanol, and 400 ml of distilled water, and the whole wasadjusted to pH 2 with phosphoric acid. With introduction of nitrogenthereinto, the solution was heated to 70° C. After 1.5 hours ofreaction, the solution was adjusted to pH 7.0 with sodium hydroxide, theresulting salts were adsorbed onto an adsorbent “KYOWAAD 1000”(manufactured by Kyowa Chemical Industry Co., Ltd.), and the solvent wasremoved to obtain 3-benzyloxy-1,2-propanediol.

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) 3.50-3.71 (4H, m, CH₂ OH,CH₂ O—CH₂Ph), 3.86-3.91 (1H, m, CHOH), 4.54 (2H, m, —CH₂ Ph) 7.27-7.38(5H, m, —CH₂ Ph).

Example 1-3

To a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 27.3 g (0.15 mol) of3-benzyloxy-1,2-propanediol, 1.27 g of dry toluene, and 0.9 g (39 mmol:26 mol %) of sodium. With introduction of nitrogen thereinto, the wholewas stirred at room temperature until sodium dissolved. The solution wascharged into a 5 L autoclave and the atmosphere was replaced bynitrogen, followed by heating to 100° C. Then, 1473 g (33.5 mol) ofethylene oxide was added thereto at 100 to 150° C. under a pressure of 1MPa or lower, followed by continuation of the reaction for another 1hour. Unreacted ethylene oxide gas was removed under reduced pressure,then the whole was cooled to 60° C. and adjusted to pH 7.5 with 85%aqueous phosphoric acid solution to obtain the following compound (p1).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.40-3.80 (901H, m, —CH₂O(CH₂CH₂ O)_(m)H, CHO(CH₂CH₂ O)_(m)H, CH₂ OCH₂Ph), 4.54 (2H, s, —CH₂Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn): 9978,weight average molecular weight (Mw): 10171, polydispersity (Mw/Mn):1.019, peak top molecular weight (mp): 10044;<whole peak> number average molecular weight (Mn): 9865, weight averagemolecular weight (Mw): 10114, polydispersity (Mw/Mn): 1.025, peak topmolecular weight (mp): 10044.

Example 1-4

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 100 g (10 mmol) of the above compound (p1) and 320 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of water. After cooling to room temperature, 10.12 g (100 mmol)of triethylamine and 6.87 g (60 mmol) of methanesulfonyl chloride wereadded thereto, followed by 6 hours of reaction at 40° C. The reactionliquid was filtered and the filtrate was transferred into a 500 mlround-bottom flask fitted with a thermometer, a nitrogen-introducingtube, a stirrer, and a condenser tube. Then, 19.3 g (100 mmol) of 28%methanol solution of sodium methoxide was added thereto, followed by 6hours of reaction at 70° C. Subsequently, 27 g of an adsorbent “KYOWAAD700” (manufactured by Kyowa Chemical Industry Co., Ltd.) was added tothe reaction liquid and the whole was further stirred at 70° C. for 1hour to adsorb excessive sodium methoxide. After filtration of thereaction liquid, the filtrate was charged into a 1. L beaker andcrystallization was carried out by adding 300 g of ethyl acetate and 350g of hexane. The precipitated crystals were collected into a 1 L beakerby filtration and dissolved under heating at 40° C. with adding 400 g ofethyl acetate. Thereafter, 300 g of hexane was added and crystallizationwas again carried out. The precipitated crystals were collected byfiltration and dried to obtain the following compound (p2).

¹H-NMR (CDCl₃, internal standard; TMS) δ (ppm); 3.38 (6H, s, —CH₃ ),3.40-3.80 (901H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OCH₂Ph), 4.54 (2H, s, —CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn) 10320,weight average molecular weight (Mw): 10551, polydispersity (Mw/Mn):1.022, peak top molecular weight (MP) 10390; <whole peak> number averagemolecular weight (Mn); 10128, weight average molecular weight (Mw);10452, polydispersity (Mw/Mn): 1.032, peak top molecular weight (Mp):10390.

Example 1-5

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added 15g of the above compound (p2), and 15 g of 5% palladium-carbon (50%hydrous product). After the replacement by nitrogen, 300 ml of methanoland 150 ml of cyclohexene were added thereto and the whole was heated togentle reflux at 52 to 55° C. to allow to react for 5 hours. Aftercooling of the reaction mixture to room temperature, thepalladium-carbon was removed by filtration and the filtrate wasconcentrated. The concentrate was crystallized by adding 50 ml of ethylacetate and 50 ml of hexane. The resulting crystals were collected byfiltration and dried to obtain the following compound (p3).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.38 (6H, s, —CH₃ ),3.40-3.80 (901H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OH).

GPC analysis: <main peak> number average molecular weight (Mn): 10069,weight average molecular weight (Mw): 10227, polydispersity (Mw/Mn):1.016, peak top molecular weight (Mp): 103511<whole peak> number averagemolecular weight (Mn): 9860, weight average molecular weight (Mw):10294, polydispersity (Mw/Mn): 1.044, peak top molecular weight (Mp):10351.

Example 2 Synthesis of Mesylate Compound (Group I(b), Y═CH₃) (Case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 10000)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 20 g (2 mmol) of the above compound (p3) and 75 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of water. After cooling to room temperature, 1.012 g (10 mmol)of triethylamine and 0.687 g (6 mmol) of methanesulfonyl chloride wereadded thereto, followed by 6 hours of reaction at 40° C. and another 1hour of reaction at 50° C. The reaction liquid was filtered and 1.0 g ofan adsorbent “KYOWAAD 1000” (manufactured by Kyowa Chemical IndustryCo., Ltd.) was added to the filtrate and the whole was further stirredat 60° C. for 1 hour to adsorb triethylamine salt of methanesulfonicacid as a by-product. After filtration of the reaction liquid, thefiltrate was charged into a 500 ml beaker and crystallization wascarried out by adding 100 ml of ethyl acetate and 150 ml of hexane. Theprecipitated crystals were collected into a 300 ml beaker by filtrationand dissolved under heating at 40° C. with adding 100 ml of ethylacetate. Thereafter, 100 ml of hexane was added and crystallization wasagain carried out. The precipitated crystals were collected byfiltration and dried to obtain the following mesylate compound (p4).

¹H-NMR (CDCl₃, internal standards TMS) δ (ppm): 3.08 (3H, s, —SO₃ CH₃ ),3.38 (6H, s, —CH₃ ), 3.40-3.80 (899H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃,CHO(CH₂CH₂ O)_(m)CH₃), 4.27-4.44 (2H, m, —CH₂ OSO₃CH₃.

GPC analysis: <main peak> number average molecular weight (Mn): 10054,weight average molecular weight (Mw): 10214, polydispersity (Mw/Mn):1.016, peak top molecular weight (Mp): 10442; <whole peak> numberaverage molecular weight (Mn): 9778, weight average molecular weight(Mw): 10252, polydispersity (Mw/Mn): 1.049, peak top molecular weight(Mp): 10442.

Example 3 Synthesis of Amino Compound (Group II(j)) (Case of R=MethylGroup, A¹O, A²O=Oxyethylene Group, n=0, and Molecular Weight=about10000)

Into a 100 ml round-bottom flask fitted with a thermometer, a stirrer,and a condenser tubs were charged 1 g (0.1 mmol) of the above mesylatecompound (p4) and 50 ml of 28% aqueous ammonia, and the whole wasstirred at 50° C. for 36 hours. The liquid temperature was raised to 65°C. and ammonia was removed with introduction of nitrogen thereinto for 2hours. After cooling to room temperature, 10 g of sodium chloride wasadded thereto, followed by extraction with 10 ml of chloroform threetimes. The resulting chloroform layer was dried over sodium sulfate andafter filtration, chloroform was removed by evaporation. Then, 100 ml ofhexane was added to the resulting concentrate to effect reprecipitation.The precipitated crystals were collected by filtration and dried toobtain the following compound (p5).

¹H-NMR (D₂O, internal standard: H₂O 4.7 ppm) δ (ppm): 3.38 (6H, s, —CH₃), 2.93-3.11 (2H, m, —CH₂ NH₂), 3.40-3.80 (899H, m, —CH₂ O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃).

Example 4 Synthesis of Aldehyde Compound (Group I(f)) (Case of R=MethylGroup, A¹O, A²O=Oxyethylene Group, n=0, and Molecular Weight=about10000) Example 4-1

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 10 g (1. mmol) of the above mesylate compound (p4) and40 ml of toluene, and the whole was heated under reflux to effectazeotropic removal of water, followed by cooling to room temperature. Onthe other hand, into a 100 ml round-bottom flask fitted with athermometer, a nitrogen-introducing tube, a stirrer, a Dean-stark tube,and a condenser tube were added 14.8 g (0.1 mol) of3,3-diethoxy-1-propanol and 40 ml of toluene, and the whole was heatedunder reflux to effect azeotropic removal of water. After cooling toroom temperature, 0.36 g (15.6 mmol) of sodium was added and the wholewas stirred at room temperature for 2 hours until it was dissolved.After dissolution of sodium was confirmed, the reaction liquid waspoured into the round-bottom flask containing the compound (p4) fromwhich water had been removed as above, followed by 12 hours of reactionat 110° C. After cooling of the reaction liquid to 40° C., 0.36 g (20mmol) of ion-exchange water was added and the whole was stirred for 30minutes. Then, 50 ml of 20% aqueous sodium chloride solution was addedthereto and the aqueous layer was adjusted to pH 7.0 with 85% phosphoricacid. After the upper toluene layer was separated, the aqueous layer wasextracted twice with chloroform. The toluene layer and the chloroformlayer were combined and dried over sodium sulfate. After filtration,toluene and chloroform were removed by evaporation to effectconcentration. The concentrate was dissolved under heating with adding50 ml of ethyl acetate and then 50 ml of hexane was added to precipitatecrystals. The resulting crystals were collected by filtration anddissolved under beating by adding 50 ml of ethyl acetate and then 50 mlof hexane was added to precipitate crystals again. This reprecipitationoperation was repeated three times. Thereafter, the precipitatedcrystals were collected by filtration and dried to obtain the followingacetal compound (p6).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 1.20 (6H, t,—CH₂CH₂CH(OCH₂ CH₃ )₂), 1.88-1.92 (2H, m, —CH₃ CH₂ CH(OCH₂CH₃)₂), 3.38(6H, s, —CH₃ ), 3.40-3.80 (907H, at, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂O)_(m)CH₃, —CH₂ —O—CH₂ CH₂CH(OCH₂ CH₃)₂), 4.64 (1H, t, —CH₂CH₂CH(OCH₂CH₃)₂).

GPC analysis: <main peak> number average molecular weight (Mn): 9898,weight average molecular weight (Mw): 10076, polydispersity (Mw/Mn):1.018, peak top molecular weight (Mp): 10215; <whole peak> numberaverage molecular weight (Mn): 9297, weight average molecular weight(Mw): 9932, polydispersity (Mw/Mn): 1.068, peak top molecular weight(Mp): 10215.

Example 4-2

Into a 200 ml beaker was weighed 4 g of the resulting acetal compound(p6). Then, 80 g of ion-exchange water was added to dissolve thecrystals and the solution was adjusted to pH 1.5 with 85% phosphoricacid, followed by 2 hours of stirring at room temperature. Thereafter,16 g of sodium chloride was added and dissolved and the whole wasadjusted to pH 7.0 with 30% aqueous sodium hydroxide solution, followedby extraction with chloroform. The resulting chloroform layer was driedover sodium sulfate and after filtration, chloroform was removed byevaporation to effect concentration. The concentrate was dissolved underheating by adding 30 ml of toluene and 30 ml of ethyl acetate and then60 ml of hexane was added to precipitate crystals, which was collectedby filtration. The resulting crystals were weighed into a 200 ml beakerand dissolved under heating by adding 30 ml of toluene and 30 ml ofethyl acetate and then 60 ml of hexane was added to precipitate crystalsagain, which was collected by filtration and dried to obtain thefollowing aldehyde compound (p7).

¹H-NMR (CDCl₃, internal standards TMS) δ (ppm); 2.65 (2H, m, CH₂ COH),3.38 (6H, s, —CH₃ ), 3.40-3.80 (903H, m, —CH₂ O(CH₂CH₂ O)_(m),CHO(CH₂CH₂ O)_(m), CH₂ OCH₂ CH₂COH), 9.78 (1H, m, CH₂COH).

Example 5

Into 50 ml of 100 mM sodium dihydrogen phosphate was added and dissolved63 mg (20 mM) of sodium cyanotrihydroborate. To 1 ml of the solutionwere added 5.0 mg (0.1 μmol) of OVA (ALUBUMIN, CHIKEN EGG, molecularweight about 40000) and 100 mg of the aldehyde compound (p7), followedby 12 hours of stirring at room temperature. The reaction liquid wasdiluted five times with ion-exchange water and 20 μl of the dilutedsolution was mixed with 20 μl of a Tris-SDS sample-treating liquid,followed by 2.5 minutes of heating on a boiling water bath. The treatedliquid was analyzed by a sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (4 to 20%). The gel was stained by CBS staining. Theresults were shown in FIG. 1. (A) is a lane of OVA+aldehyde compound,(B) a lane of OVA alone, and (C) a lane of a marker (Bio-rad Broad rangeSDS-PAGE standards) which shows bands of molecular weights of 201000,130000, 94000, 48600, 36400, 29800, 20600, and 600 from the top.

From these results, a band of the starting OVA did not remain but bandsof molecular weights corresponding to the cases that OVA was modifiedwith the compound (p6) at 1 to 15 places per one molecule were observedin (A).

Example 6

For evaluating stability of the compounds of the invention, thefollowing model compound was synthesized and the stability was compared.

Example 6-1

In 50 ml of methanol was dissolved 63 mg (20 mM) of sodiumcyanotrihydroborate. Into 2 ml of the solution were added 0.5 g of thealdehyde compound (p7) and 50 μl of n-butylamine, followed by 18 hoursof stirring at room temperature. Methanol was removed by evaporation toeffect concentration and then the concentrate was extracted by adding 20ml of chloroform and 20 ml of 20% aqueous sodium chloride. Theextraction operation was repeated three times. The resulting chloroformlayer was dried over sodium sulfate and after filtration, concentrated.The resulting concentrate was dissolved by adding 20 ml of ethyl acetateand then 30 ml of hexane was added to precipitate crystals, which wascollected by filtration. The resulting crystals were weighed into a 1.00ml beaker and dissolved under heating with adding 20 ml of ethyl acetateand then 20 ml of hexane was added to precipitate crystals again, whichwas collected by filtration and dried to obtain the following compound(p8).

Example 6-2 Evaluation of Stability (Accelerated Aging Test)

The synthesized above compound (p8) was weighed in an amount of 12 mg,and 1 ml of 100 mM phosphate buffer (pH=8.8) was added thereto, followedby 12 hours of stirring on a water bath at 75° C. GPC measurement wascarried out before starting and after completion of stirring. Theresults are shown in FIG. 2 and FIG. 3. FIG. 2 is a GPC chart of thesample before starting and FIG. 3 is a GPC chart of the sample of (p8)after heating.

Comparative Example 1

The following compound (p9) having a molecular weight of about 10700purchased from Shearwater Polymers, Inc. was weighed in an amount of 107mg, and 10 μl of n-butylamine and 1 ml of chloroform were added thereto,followed by 18 hours of stirring at room temperature. Chloroform wasremoved by evaporation to effect concentration and then the concentratewas dissolved under heating with adding 20 ml of ethyl acetate and then30 ml of hexane was added to precipitate crystals, which was collectedby filtration. The resulting crystals were weighed into a 100 ml beakerand dissolved under heating with adding 20 ml ethyl acetate and then 20ml of hexane was added to precipitate crystals again, which wascollected by filtration and dried to obtain the following compound(p10).

Using the above synthesized compound (p10), the same operations as inExample 6-2 were carried out and GPC measurement was conducted. Theresults are shown in FIG. 4 and FIG. 5. FIG. 4 is a GPC chart of thesample of (p10) before starting and FIG. 5 is a GPC chart of the sampleof (p10) after heating.

From the results of FIG. 2 and FIG. 3, the compounds of the inventiondid not hydrolyzed and exhibited a high stability. On the other hand,from the results of FIG. 4 and FIG. 5, a compound having ½ molecularweight was formed in an amount of about 25% in the case of ComparativeExample, (p10), which showed that the urethane bond was cleaved and thebranched polyethylene glycol was decomposed into a single chain.

Example 7 Synthesis of Compound (p) (Case of R=Methyl Group, A¹O,A²O=Oxyethylene Group, n=0, and Molecular Weight=about 1.9000) Example7-1

In a similar manner to Example 1-3, 2850 g (64.8 mol) of ethylene oxidewas charged and the following compound (p11) was obtained.

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.40-3.80 (1733H, m,—CH₂ O(CH₂CH₂ O)_(m)H, CHO(CH₂CH₂ O)_(m) H, CH₂ OCH₂Ph), 4.54 (2H, s,—CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn): 18521,weight average molecular weight (Mw): 18758, polydispersity (Mw/Mn):1.013, peak top molecular weight (Mp): 19108;<whole peak> number average molecular weight (Mn): 18403, weight averagemolecular weight (Mw): 1891.3, polydispersity (Mw/Mn): 1.028, peak topmolecular weight (Mp): 19108.

Example 7-2

In a similar manner to Example 1-4, the following compound (p12) wasobtained using 100 g (5 mmol) of (p11), 320 g of toluene, 5.06 g (50mmol) of triethylamine, 3.44 g (30 mmol) of methanesulfonyl chloride,and 9.65 g (50 mmol) of 28% methanol solution of sodium methoxide.

¹H-NMR (CDCl₃, internal standard: TMS) δ (Ppm) 3.38 (6H, s, —CH₃ ),3.40-3.80 (1733H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OCH₂Ph), 4.54 (2H, s, —CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn): 18365,weight average molecular weight (Mw); 18602, polydispersity (Mw/Mn):1.013, peak top molecular weight (Mp): 18992;<whole peak> number average molecular weight (Mn); 18290, weight averagemolecular weight (Mw): 18861, polydispersity (Mw/Mn): 1.031, peak topmolecular weight (Mp): 18992.

Example 7-3

The following compound (p13) was obtained in a similar manner to Example1-5.

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.38 (6H, s, —CH₃ ),3.40-3.80 (1733H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃,CH₂OH.

GPC analysis: <main peak> number average molecular weight (Mn): 18395,weight average molecular weight (Mw): 18632, polydispersity (Mw/Mn):1.013, peak top molecular weight (Mp): 18989;<whole peak> number average molecular weight (M11): 18146, weightaverage molecular weight (Mw): 18750, polydispersity (Mw/Mn): 1.033,peak top molecular weight (MD): 18989.

Example 8 Synthesis of Carboxyl Compound (Group II(k)) and SuccinimideEster Compound (Group I(a)) (Case of R=Methyl Group, A¹O,A²O=Oxyethylene Group, n=0, and Molecular Weight=about 19000)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 20 g (1.0 mmol) of the above compound (p13), 50 mg ofsodium acetate, and 100 ml of toluene, and the whole was heated underreflux to effect azeotropic removal of water. Then, 137 mg (1.2 mmol) ofglutaric anhydride was added to the reaction liquid, followed by 12hours of reaction at 105° C. After completion of the reaction, thereaction liquid was cooled to 40° C. and 150 mg (1.3 mmol) ofN-hydroxysuccinimide and 289 mg (1.4 mmol) of dicyclohexylcarbodiimidewere added thereto, followed by 6 hours of reaction. The reaction liquidwas filtered to remove precipitated urea and, after addition of 50 ml ofethyl acetate to the filtrate, 150 ml of hexane was added to precipitatecrystals. The precipitated crystals were collected by filtration anddissolved under heating with adding 100 ml of ethyl acetate. Then, 100ml of hexane was added thereto to crystallize the product again. Theprecipitated crystals were collected by filtration and dried to obtainthe following succinimide ester compound (p14).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 2.07 (2H, m, —OCOCH₂CH₂CH₂ COON—), 2.50 (2H, t, —OCOCH₂ CH₂CH₂COON—), 2.72 (2H, t, —OCOCH₃CH₂CH₂ COON—), 2.84 (4H, s, succinimide) 3.38 (6H, s, —CH₃ ), 3.40-3.80(1731H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃), 4.10-4.30(2H, m, —CH₂ OCOCH₂CH₂CH₂COON—).

Example 9 Synthesis of p-Nitrophenyl Carbonate Compound (Group I(d))(Case of R=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 19000)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 20 g (1.0 mmol) of the above compound (p13) and 100 mlof toluene, and the whole was heated under reflux to effect azeotropicremoval of water. The reaction liquid was cooled to 80° C. andtriethylamine and p-nitrophenyl chloroformate were added to thereto,followed by 5 hours of reaction at 80° C. After completion of thereaction, the reaction liquid was filtered and, after addition of 100 mlof ethyl acetate to the filtrate, 200 ml of hexane was added toprecipitate crystals. The precipitated crystals were collected byfiltration and dissolved under heating with adding 100 ml of ethylacetate. Then, 100 ml of hexane was added thereto to crystallize theproduct again. The crystallization operation was repeated five times intotal. The crystals collected by filtration were dried to obtain thefollowing p-nitrophenyl carbonate compound (p15).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) s 3.38 (6H, s, —CH₃ ),3.40-3.80 (1731H, m, —CH₂—O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃)4.30-4.50 (2H, m, —CH₂ OCOOPhNO₂), 7.39 (2H, d, -PhNO₂), 8.28 (2H, d,-PhNO₂).

Example 10 Synthesis of Compound (p) (Case of R=Methyl Group, A¹O,A²O=Oxyethylene Group, n=about 15, and Molecular Weight=about 19500)

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 67 g (3.5 mmol) of the compound (p13) obtained inExample 7-3 and 400 ml of toluene, and the whole was heated under refluxto effect azeotropic removal of water. The reaction liquid was cooled to40° C. and 0.41 g (2.1 mmol) of a 28% methanol solution of sodiummethoxide was added thereto. After heating to 70° C., about 200 ml of amixed solution of toluene-methanol was removed by evaporation withnitrogen bubbling. The solution was charged into a 5 L autoclave and theatmosphere was replaced by nitrogen, followed by heating to 100° C.Then, 9.2 g (0.2 mol) of ethylene oxide was added thereto at 100 to 150°C. under a pressure of 1 MPa or lower, followed by continuation of thereaction for another 3 hours. Unreacted ethylene oxide gas and toluenewere removed under reduced pressure, then the whole was cooled to 60° C.and adjusted to pH 7.5 with a 85% aqueous phosphoric acid solution toobtain the following compound (p16). ¹H-NMR (CDCl₃, internal standard;TMS) δ (ppm) a 3.38 (6H, s, —CH₃ ), 3.40-3.80 (1853H, m, —CH₂ O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, —CH₂ O(CH₂CH₂ O)_(n)H).

GPC analysis: <main peak> number average molecular weight (Mn): 19153,weight average molecular weight (Mw): 19462, polydispersity (Mw/Mn):1.016, peak top molecular weight (Mp): 19612,<whole peak> number average molecular weight (Mn): 18473, weight averagemolecular weight (Mw): 19087, polydispersity (Mw/Mn): 1.033, peak topmolecular weight (14p) 119612.

Example 11 Synthesis of Mesylate Compound (Group I(b), Y=CH₃) (Case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=about 15, and MolecularWeight=about 19500)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 10 g (0.5 mmol) of the above compound (p16) and 75 gof toluene, and the whole was heated under reflux to effect azeotropicremoval of water. After cooling to room temperature, 0.253 g (2.5 mmol)of triethylamine and 0.172 g (1.5 mmol) of methanesulfonyl chloride wereadded thereto, followed by 6 hours of reaction at 40° C. and another 1hour of reaction at 50° C. The reaction liquid was filtered and 0.5 g ofan adsorbent “KYOWAAD 1000” was added to the filtrate and the whole wasstirred at 60° C. for 1 hour to adsorb triethylamine salt ofmethanesulfonic acid as a by-product. After filtration of the reactionliquid, the filtrate was charged into a 300 ml beaker andcrystallization was carried out with adding 50 ml of ethyl acetate and70 ml of hexane. The precipitated crystals were collected into a 300 mlbeaker by filtration and dissolved under heating at 40° C. with adding50 ml of ethyl acetate. Thereafter, 50 ml of hexane was added andcrystallization was again carried out. The precipitated crystals werecollected by filtration and dried to obtain the following mesylatecompound (p17).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) 3.08 (3H, s, —SO₃ CH₃ ),3.38 (6H, s, —CH₃ ), 3.40-3.80 (1851H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃,CHO(CH₂CH₂ O)_(m)CH₃, —CH₂ O(CH₂CH₂ O)_(n)SOOCH₃), 4.37-4.39 (2H, m—CH₂—O—(CH₂CH₂O)_(n-1)CH₂ CH₂ OSOOCH₃).

GPC analysis: <main peak> number average molecular weight (Mn): 19253,weight average molecular weight (Mw): 19601, polydispersity (Mw/Mn):1.018, peak top molecular weight (Mp): 19770;<whole peak> number average molecular weight (Mn): 18400, weight averagemolecular weight (Mw): 19140, polydispersity (Mw/Mn): 1.040, peak topmolecular weight (MP): 19770.

Example 12 Synthesis of Aldehyde Compound (Group I(f)) (Case of R=MethylGroup, A¹O, A²O=Oxyethylene Group, n=about 15, and MolecularWeight=about 19500) Example 12-1

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 10 g (0.5 mmol) of the above mesylate compound (p17) and40 ml of toluene, and the whole was heated under reflux to effectazeotropic removal of water, followed by cooling to room temperature. Onthe other hand, into a 100 ml round-bottom flask fitted with athermometer, a nitrogen-introducing tube, a stirrer, a Dean-stark tube,and a condenser tube were added 7.4 g (50 mmol) of3,3-diethoxy-1-propanol and 40 ml of toluene, and the whole was heatedunder reflux to effect azeotropic removal of water. After cooling toroom temperature, 0.17 g (7.4 mmol) of sodium was added and the wholewas stirred at room temperature for 2 hours until it was dissolved.After dissolution of sodium was confirmed, the reaction liquid waspoured into the round-bottom flask containing the compound (p17) fromwhich water had been removed as above, followed by 4 hours of reactionat 70° C. After cooling of the reaction liquid to 40° C., 0.18 g (10mmol) of ion-exchange water was added and the whole was stirred for 30minutes. Then, 30 ml of 20% aqueous sodium chloride solution was addedthereto and the aqueous layer was adjusted to pH 7.0 with 85% aphosphoric acid. After the upper toluene layer was separated, theaqueous layer was extracted twice with chloroform. The toluene layer andthe chloroform layer were combined and dried over sodium sulfate. Afterfiltration, toluene and chloroform were removed by evaporation to effectconcentration. The concentrate was dissolved under heating with adding50 ml of ethyl acetate and then 50 ml of hexane was added to precipitatecrystals. The resulting crystals were collected by filtration and thendissolved under heating with adding 50 ml of ethyl acetate and then 50ml of hexane was added to precipitate crystals again. Thisreprecipitation operation was repeated three times. Thereafter, theprecipitated crystals were collected by filtration and dried to obtainthe following acetal compound (p18).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 1.20 (6H, t,—CH₂CH₂CH(OCH₂ CH₃ )₂), 1.88-1.92 (2H, m, —CH₂ CH₂ CH(OCH₂CH₃)₂) 3.38(6H, s, —CH₃ ), 3.40-3.80 (1857H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂O)_(m)CH₃, —CH₂ O(CH₂CH₂ O)_(n) CH₂ CH₂CH(OCH₂ CH₃)₂), 4.64 (1H, t,—CH₂CH₂ CH(OCH₂CH₃)₂).

GPC analysis: <main peak> number average molecular weight (Mn): 19318,weight average molecular weight (Mw) 19699, polydispersity (Mw/Mn)1.020, peak top molecular weight (Mp) 197707<whole peak> number averagemolecular weight (Mn): 3.8302, weight average molecular weight (Mw):3.9168, polydispersity (Mw/Mn): 1.047, peak top molecular weight (MP):19770.

Example 3.2-2

Into a 100 ml beaker was weighed 2 g of the resulting acetal compound(p18). Then, 40 g of ion-exchange water was added to dissolve thecrystals and the solution was adjusted to pH 1.5 with 85% phosphoricacid, followed by 2 hours of stirring at room temperature. Thereafter, 8g of sodium chloride was added and dissolved and the whole was adjustedto pH 7.0 with 30% aqueous sodium hydroxide solution, followed byextraction with chloroform three times. The resulting chloroform layerwas dried over sodium sulfate and after filtration, chloroform wasremoved by evaporation to effect concentration. The concentrate wasdissolved under heating with adding 30 ml of toluene and 30 ml of ethylacetate and then 60 ml of hexane was added to precipitate crystals,which was collected by filtration. The resulting crystals were weighedinto a 200 ml beaker and dissolved under heating with adding 30 ml oftoluene and 30 ml of ethyl acetate and then 60 ml of hexane was added toprecipitate crystals again, which was collected by filtration and driedto obtain the following aldehyde compound (p19).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 2.66-2.69 (2H, m, CH₂COH), 3.38 (6H, s, —CH₃ ), 3.40-3.80 (1855H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃,CHO(CH₂CH₂ O)_(m)CH₃,

—CH₂ O(CH₂CH₂ O)_(n) CH₂ CH₂COH), 9.79 (1H, t, —CH₂CH₂COH).

Example 13 Synthesis of Mesylate Compound (Group I(b), Y=CH₃) (case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 19000)

Using the compound (p13) as a starting material, the following mesylatecompound (p20) was obtained in a similar manner to Example 2.

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.08 (3H, s, —SO₃ CH₃ ),3.38 (6H, s, —CH₃ ), 3.40-3.80 (1731H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃,CHO(CH₂CH₂ O)_(m)CH₃), 4.27-4.44 (2H, m, —CH₂ OSO₃CH₃).

GPC analysis: <main peak> number average molecular weight (Mn): 18435,weight average molecular weight (Mw): 18682, polydispersity (Mw/Mn):1.013, peak top molecular weight (Mp): 18740;<whole peak> number average molecular weight (Mn): 18081, weight averagemolecular weight (Mw): 18721, polydispersity (Mw/Mn): 1.035, peak topmolecular weight (Mp): 18740.

Example 14 Synthesis of Amino Compound (Group II(j)) (Case of R=MethylGroup, A¹O, A²O=Oxyethylene Group, n=0, and Molecular Weight=about19000)

Using the compound (p20) as a starting material, the following aminocompound (p21) was obtained in a similar manner to Example 3.

¹H-MMR (D₂O, internal standard: H₂O=4.7 ppm) δ (ppm): 3.38 (6H, s, —CH₃), 2.93-3.11 (2H, m, —CH₂ NH₂), 3.40-3.80 (1731H, m, —CH₂ O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃).

Example 15 Synthesis of Maleimide Compound (Group I(e)) (Case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 19000)

Into a 100 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were charged7.5 g (0.35 mmol) of the above compound (p21), 35 ml of ethyl acetate,and 73 μl of triethylamine, and the whole was heated at 45° C. todissolve them. Then, 0.14 g (0.525 mmol) of N-succinimidyl3-maleimidopropionate was added thereto, followed by 4 hours of reactionat 45° C. After completion of the reaction, 0.5 g of an adsorbent“KYOWAAD 700” and 0.5 g of “KYOWAAD 1000” were added thereto and thewhole was stirred at 45° C. for another 1 hour. The reaction liquid wasfiltrated and 50 ml of hexane was added to the filtrate to precipitatecrystals, which was collected by filtration. The resulting crystals wereweighed into a 200 ml beaker and dissolved under heating with adding 50ml of ethyl acetate. Then, 50 ml of hexane was added to precipitatecrystals again, which were collected by filtration and dried to obtainthe following maleimide compound (p22).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) 2.51 (2H, t, —NHCOCH₂CH₂), 3.38 (6H, s, —CH₃ ), 3.40-3.80 (1735H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃,CHO(CH₂CH₂ O)_(m)CH₃, CH₂ NHCOCH₂ CH₂ ) 6.69 (2H, s, CH═CH), 6.86 (1H,t, CH₂ NHCOCH₂CH₂.

GPC analysis: <main peak> number average molecular weight (Mn): 18425,weight average molecular weight (Mw): 18672, polydispersity (Mw/Mn):1.013, peak top molecular weight (Mp): 18742;<whole peak> number average molecular weight (Mn): 17924, weight averagemolecular weight (Mw): 19086, polydispersity (Mw/Mn): 1.065, peak topmolecular weight (Mp): 18742.

Example 16 Synthesis of Compound (p) (Case of R=Methyl Group, A¹O,A²O=Oxyethylene Group, n=0, and Molecular Weight=about 20000, about45000) Example 16-1

To a 1000 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 132.2 g (1.0 mol) of2,2-dimethyl-1,3-dioxolane-4-methanol, 202.5 g (1.05 mol) of a 28%methanol solution of sodium methoxide, and 500 ml of toluene. Withintroduction of nitrogen thereinto, toluene was refluxed under reducedpressure for 1 hour to remove the methanol by evaporation. Withmaintaining the solution at 80° C., 126.6 g (1.0 mol) of benzyl chloridewas added dropwise over a period of 2 hours using a dropping funnel,followed by another 2 hours of reaction. After completion of thereaction, the temperature was lowered to 60° C. and 10 g of KYOWAAD 600was added, followed by 1 hour of stirring. After filtration of thereaction liquid, the solvent was removed and the residue was purified bydistillation (b.p. 93-95° C./266 Pa) to obtain4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 1.36, 1.42 (3H, 3H, s,C(CH₃ )₂), 3.45-3.57 (2H, m, CH₂ O—C(CH₃)₂), 3.73-3.76 (1H, m,CHO—C(CH₃)₂), 4.03-4.07, 4.28-4.32 (2H, m, CH₂ O—CH₂Ph), 4.57 (2H, q,—CH₂ Ph), 7.15-7.40 (5H, m, —CH₂ Ph) (Ph represents a phenyl group).

Example 16-2

To 222 g (1.0 mol) of 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane wasadded 400 g of distilled water, and the whole was adjusted to pH 2 withphosphoric acid. With introduction of nitrogen thereinto, the solutionwas heated to 70° C. After 2 hours of reaction, the solution wasadjusted to pH 7.0 with sodium hydroxide. Thereto was charged 1 L ofchloroform and extraction was carried out. Then, the chloroform layerwas dried over magnesium sulfate and concentrated. Thereafter, theconcentrate was filtrated to remove salts, and thereby3-benzyloxy-1,2-propanediol was obtained. The NMR data thereof are thesame as those in Example 1-2.

Example 16-3

To a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a pressure-reducing line wereadded 27.3 g (0.15 mol) of 3-benzyloxy-1,2-propanediol, 200 g of drytoluene, and 0.77 g (33.4 mmol s 22.3 mol %) of sodium. Withintroduction of nitrogen thereinto, the temperature was raised to 35° C.to dissolve sodium. The solution was charged into a 5 L autoclave whichhad been thoroughly dried beforehand and the atmosphere was replaced bynitrogen, followed by heating to 100° C. Then, 3090 g of ethylene oxidewas introduced under pressure thereto at 100 to 150° C. under a pressureof 1 MPa or lower, followed by continuation of the reaction for another1.5 hours. Unreacted ethylene oxide gas and toluene were removed underreduced pressure, then the whole was cooled to 70° C. Then, 2.0 kg ofthe reaction liquid was taken out of the autoclave and the reactionliquid taken out was adjusted to pH 7.5 with 85% aqueous phosphoric acidsolution to obtain the following compound (p23).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.40-3.80 (1773H, m,—CH₂ O(CH₂CH₂ O)_(m)H, CHO(CH₂CH₂ O)_(m)H, CH₂ OCH₂Ph), 4.54 (2H, s,—CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn) 18920,weight average molecular weight (Mw): 19154, polydispersity (Mw/Mn):1.012, peak top molecular weight (Mp): 19639;<whole peak> number average molecular weight (Mn) 18777, weight averagemolecular weight (Mw): 19086, polydispersity (Mw/Mn): 1.017, peak topmolecular weight (Mp): 19639.

Example 16-4

Into a 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 200 g (10 mmol) of the above compound (p23) and 1000 gof toluene, and the whole was heated under reflux to effect azeotropicremoval of 200 g of toluene and water. After cooling to roomtemperature, 10.12 g (100 mmol) of triethylamine was added and the wholewas heated to 40° C. Then, 6.87 g (60 mmol) of methanesulfonyl chloridewas added thereto, followed by 3 hours of reaction at 40° C. Aftercompletion of the reaction, 19.28 g (100 mmol) of a 28% methanolsolution of sodium methoxide was added to the reaction liquid, followedby 3 hours of reaction at 40° C. The pressure was reduced withmaintaining the reaction liquid at 40° C. to remove about 200 g of amixed solution of methanol/toluene by evaporation and then salts wereremoved by filtration. Then, 500 g of toluene was added to the filtrateand the mixture was transferred into a 2 L round-bottom flask fittedwith a thermometer, a nitrogen-introducing tube, a stirrer, a Dean-starktube, and a condenser tube. The whole was heated under reflux to effectazeotropic removal of 200 g of toluene and water. After cooling to roomtemperature, 10.12 g (100 mmol) of triethylamine was added and the wholewas heated to 40° C. Then, 8.89 g (60 mmol) of methanesulfonyl chloridewas added thereto, followed by 3 hours of reaction at 40° C. Aftercompletion of the reaction, 19.28 g (100 mmol) of a 28% methanolsolution of sodium methoxide was added to the reaction liquid, followedby 3 hours of reaction at 40° C. The pressure was reduced withmaintaining the reaction liquid at 40° C. to remove about 200 g of amixed solution of methanol/toluene by evaporation and then salts wereremoved by filtration. The filtrate was heated to 50° C. and then 200 gof 25% aqueous sodium chloride was added thereto. After stirring, thewhole was left on standing to be separated into layers and the loweraqueous layer was removed. The operation of washing with water wasrepeated twice. The upper toluene layer was dried over magnesium sulfateand then filtrated, and 1 L of ethyl acetate was added to the filtrate.Then, hexane was added thereto until crystals were precipitated. Thecrystals were collected by filtration and dried to obtain the followingcompound (p24).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.38 (6H, s, —CH₃ ),3.40-3.80 (1773H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OCH₂Ph), 4.54 (2H, s, —CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis; <main peak> number average molecular weight (Mn): 19070,weight average molecular weight (Mw): 19306, polydispersity (Mw/Mn):1.012, peak top molecular weight (Mp): 19786;<whole peak> number average molecular weight (Mn): 18911, weight averagemolecular weight (Mw): 19256, polydispersity (Mw/Mn): 1.018, peak topmolecular weight (Mp): 19786.

Example 16-5

Into a pressure filter was charged 120 g of 5% palladium-carbon (50%hydrous product, manufactured by N. E. M. Cat), and solvent substitutionwas carried out four times with 500 ml of anhydrous methanol underreplacement by nitrogen to effect removal of water from thepalladium-carbon. Into a 2 L round-bottom flask fitted with athermometer, a nitrogen-introducing tube, a stirrer, and a condensertube were added 100 g of the above compound (p24), and the whole amountof the palladium-carbon which had been subjected to solventsubstitution. After replacement by nitrogen, 1200 ml of anhydrousmethanol and 500 ml of cyclohexene were added thereto and the whole washeated to 30° C. to be allowed to react for 3.5 hours. The reactionliquid was filtrated and the water content of the filtrate was measuredby means of Karl Fisher's moisture meter and found to be 1259 ppm. Thefiltrate was concentrated and 1 L of ethyl acetate was added to theconcentrate, followed by addition of hexane until crystals wereprecipitated. The resulting crystals were collected by filtration anddried to obtain the following compound (p25).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) 3.38 (6H, s, —CH₃ ),3.40-3.80 (1773H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OH).

GPC analysis: <main peak> number average molecular weight (Mn): 18971,weight average molecular weight (Mw): 19204, polydispersity (Mw/Mn):1.012, peak top molecular weight (Mp) 19687;<whole peak> number average molecular weight (Mn): 18811, weight averagemolecular weight (Mw): 19158, polydispersity (Mw/Mn): 1.018, peak topmolecular weight (Mp) 19687.

Example 16-6

In Example 16-3, 2.0 kg of dry toluene was added to about 1 kg of thereaction liquid which remained in the autoclave. After 1.0 kg of toluenewas removed by evaporation at an autoclave temperature of 95° C. underslight reduced pressure and then the atmosphere of the autoclave wasreplaced by nitrogen. After heating to 120° C., 1260 g of ethylene oxidewas introduced under pressure thereto at 100 to 150° C. under a pressureof 1 MPa or lower, followed by continuation of the reaction for another4 hours. After completion of the reaction, the whole was cooled to 70°C. and the reaction liquid was adjusted to pH 7.5 with 85% aqueousphosphoric acid solution to obtain the following compound (p26).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) s 3.40-3.80 (4045H, in,—CH₂ O(CH₂CH₂ O)_(m)H, CHO(CH₂CH₂ O)_(m)H, CH₂ OCH₂Ph), 4.54 (2H, s,—CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn) 41830,weight average molecular weight (Mw): 42621, polydispersity (Mw/Mn):1.019, peak top molecular weight (Mp): 44594:<whole peak> number average molecular weight (Mn): 40548, weight averagemolecular weight (Mw): 42059, polydispersity (Mw/Mn): 1.037, peak topmolecular weight (Mp): 44594.

Example 16-7

Into a 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 270 g (6 mmol) of the above compound (p26) and 1000 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of 200 g of toluene and water. After cooling to roomtemperature, 6.65 g (65.7 mmol) of triethylamine was added and the wholewas heated to 40° C. Then, 4.51 g (39.4 mmol) of methanesulfonylchloride was added thereto, followed by 3 hours of reaction at 40° C.After completion of the reaction, 25.3 g (131.4 mmol) of a 28% methanolsolution of sodium methoxide was added to the reaction liquid, followedby 3 hours of reaction at 40° C. The pressure was reduced withmaintaining the reaction liquid at 40° C. to remove about 200 g of amixed solution of methanol/toluene by evaporation and then salts wereremoved by filtration. Then, 500 g of toluene was added to the filtrateand the mixture was transferred into a 2 L round-bottom flask fittedwith a thermometer, a nitrogen-introducing tube, a stirrer, a Dean-starktube, and a condenser tube. The whole was heated under reflux to effectazeotropic removal of 200 g of toluene and water. After cooling to roomtemperature, 6.65 g (65.7 mmol) of triethylamine was added and the wholewas heated to 40° C. Then, 4.51 g (39.4 mmol) of methanesulfonylchloride was again added thereto, followed by 3 hours of reaction at 40°C. After completion of the reaction, 25.3 g (131.4 mmol) of a 28%methanol solution of sodium methoxide was added to the reaction liquid,followed by 3 hours of reaction at 40° C. The pressure was reduced withmaintaining the reaction liquid at 40° C. to remove about 200 g of amixed solution of methanol/toluene by evaporation and then salts wereremoved by filtration. The filtrate was heated to 50° C. and then 200 gof 25% aqueous sodium chloride was added thereto. After stirring, thewhole was left on standing and separated into layers and the loweraqueous layer was removed. The operation of washing with water wasrepeated twice. The upper toluene layer was dried over magnesium sulfateand then filtrated, and 1 L of ethyl acetate was added to the filtrate.Then, hexane was added thereto until crystals were precipitated. Thecrystals were collected by filtration and dried to obtain the followingcompound (p27).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.38 (6H, s, —CH₃ ),3.40-3.80 (4045H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OCH₂Ph), 4.54 (211, s, —CH₂ Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

GPC analysis: <main peak> number average molecular weight (Mn): 42206,weight average molecular weight (Mw): 43056, polydispersity (Mw/Mn):1.020, peak top molecular weight (Mp): 45057;<whole peak> number average molecular weight (Mn): 40990, weight averagemolecular weight (Mw): 42519, polydispersity (Mw/Mn): 1.037, peak topmolecular weight (Mp): 45057.

Example 16-8

Into a pressure filter was charged 200 g of 5% palladium-carbon (50%hydrous product, manufactured by N. E. M. Cat.), and solventsubstitution was carried out four times with 500 ml of anhydrousmethanol under replacement by nitrogen to effect removal of water fromthe palladium-carbon. Into a 2 L round-bottom flask fitted with athermometer, a nitrogen-introducing tube, a stirrer, and a condensertube was added 100 g of the above compound (p27) and the whole amount ofthe palladium-carbon which had been subjected to solvent substitution.After replacement by nitrogen, 1200 ml of anhydrous methanol and 500 mlof cyclohexene were added thereto and the whole was heated to 30° C. tobe allowed to react for 3.5 hours. The reaction liquid was filtrated andthe water content of the filtrate was measured by means of Karl Fisher'smoisture meter and found to be 2215 ppm. The filtrate was concentratedand 1 L of ethyl acetate was added to the concentrate, followed byaddition of hexane until crystals were precipitated. The resultingcrystals were collected by filtration and dried to obtain the followingcompound (p28).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 3.38 (6H, s, —CH₃ ),3.40-3.80 (4045H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂OH).

GPC analysis: <main peak> number average molecular weight (Mn): 42121,weight average molecular weight (Mw): 42946, polydispersity (Mw/Mn):1.020, peak top molecular weight (Mp): 45057;<whole peak> number average molecular weight (Mn): 41021, weight averagemolecular weight (Mw): 42450, polydispersity (Mw/Mn): 1.035, peak topmolecular weight (Mp) 45057.

Example 17 Synthesis of Amino Compound (Group II(g)) (Case of R=MethylGroup, A¹O, A²O=Oxyethylene Group, n=0, and Molecular Weight=about45000) Example 17-1

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added 70g of the above compound (p28) and 70 g of ion-exchanged water, and thewhole was heated to 40° C. to dissolve them. After dissolution, thesolution was cooled to 10° C. or lower and 4.38 g of 50% aqueouspotassium hydroxide solution was added thereto. Subsequently, 210 g ofacrylonitrile was added dropwise over a period of 2 hours withmaintaining a temperature of 5 to 10° C. After the dropwise addition,the reaction was continued for another 2 hours and 26.25 g of 8.5%aqueous phosphoric acid solution was added dropwise, followed byneutralization. After addition of 140 g of ion-exchanged water to thereaction liquid, the mixture was transferred into a separating funneland 210 ml of ethyl acetate was added. After stirring, the whole wasleft on standing and the upper ethyl acetate layer was discarded. Theextraction with ethyl acetate was repeated six times. After completionof the extraction, 65 g of sodium chloride was added to the aqueouslayer and dissolved therein, and then the solution was extracted with280 ml of chloroform. The resulting chloroform layer was dried overmagnesium sulfate, filtrated, and then concentrated. Thereafter, 700 mlof ethyl acetate was added to the concentrate, which was dissolvedtherein. Then, hexane was added thereto until crystals wereprecipitated. The crystals were collected by filtration and againdissolved in 700 ml of ethyl acetate under heating. After cooling toroom temperature, hexane was added thereto until crystals wereprecipitated. The crystals were collected by filtration and dried toobtain the following nitrile compound (p29).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 2.59-2.66 (2H, —CH₂CH₂CN), 3.38 (6H, s, —CH₃ ), 3.40-3.80 (4047H, m, —CH₂ O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃, CH₂ OCH₂ CH₂ CN).

GPC analysis: <main peak> number average molecular weight (Mn): 41849,weight average molecular weight (Mw): 42666, polydispersity (Mw/Mn):1.020, peak top molecular weight (Mp): 44594;<whole peak> number average molecular weight (Mn): 40271, weight averagemolecular weight (Mw): 41980, polydispersity (Mw/Mn): 1.042, peak topmolecular weight (Mp) 44594.

Example 17-2

To a 1 L autoclave were added 50 g of the nitrile compound of theformula (p29), 500 g of toluene, and 4.5 g of nickel (manufactured by N.E. M. Cat., 5136p), and the whole was heated to 60° C. The autoclave waspressurized with ammonia until the inner pressure reached 0.7 MPa andthen with hydrogen until the inner pressure reached 4.5 MPa, followed by3 hours of reaction at 130° C. After the reaction, the reaction liquidwas cooled to 70° C., and purge with nitrogen was repeated until ammoniasmell disappeared. The whole amount of the reaction liquid was taken outand filtrated. After cooling of the filtrate to room temperature, hexanewas added until crystals were precipitated. The crystals were collectedby filtration and dried to obtain the following amine compound (p30).

¹H-NMR (CDCl₃, internal standards TMS) δ (ppm): 1.82-1.90 (2H, m,—CH₂CH₂ CH₂ NH₂), 2.90-2.97 (2H, m, —CH₂ CH₂ CH₂NH₂), 3.38 (6H, s, —CH₃), 3.40-3.80 (4047H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃,CH₂ OCH₂ CH₂CH₂NH₂).

Example 18 Synthesis of Maleimide Compound (Group I(e)) (Case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 45000)

Into a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added 45g (1 mmol) of the compound (p30), 42 ml of acetonitrile, and 84 ml oftoluene, and the whole was heated at 40° C. to dissolve them. Aftercooling to room temperature, 0.51 g (5 mmol) of N-methylmorpholine and399 mg (1.5 mmol) of N-succinimidyl 3-maleimidopropionate were addedthereto under light shielding, followed by 3.5 hours of reaction. Afterfiltration of the reaction liquid, 840 ml of ethyl acetate was added andhexane was added thereto until crystals were precipitated. The crystalswere collected by filtration and 42 ml of acetonitrile and 840 ml ofethyl acetate were added. After dissolution under heating, hexane wasadded until crystals were precipitate. Then, the crystals were collectedby filtration and dried to obtain the following compound (p31).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm) 1.70-1.78 (2H, m,—CH₂CH₂CH₂N), 2.45-2.53 (2H, m, —NHCOCH₂CH₂N), 3.38 (6H, s, —CH₃),3.40-3.80 (4051H, m, —CH₂O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂O)_(m)CH₃,CH₂OCH₂CH₂CH₂NHCOCH₂CH₂), 6.44 (1H, m, NHCO), 6.71 (2H, s, —CH═CH—).

GPC analysis: <main peak> number average molecular weight (Mn): 41918,weight average molecular weight (Mw): 42709, polydispersity (Mw/Mn):1.019, peak top molecular weight (Hp): 44594;<whole peak> number average molecular weight (Mn): 40231, weight averagemolecular weight (Mw): 42602, polydispersity (Mw/Mn): 1.059, peak topmolecular weight (Hp): 44594.

Example 19 Synthesis of Succinimide Compound (Group I(a)) (Case ofR=Methyl Group, A¹O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 20000)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 10 g (0.5 mmol) of the compound (p25), 0.1 g of sodiumacetate, and 100 ml of toluene, and the whole was refluxed to effectremoval of water. Then, 285 mg (2.5 mmol) of glutaric anhydride wasadded to the reaction liquid, followed by 12 hours of reaction at 110°C. After cooling of the reaction liquid, 518 mg (4.5 mmol) ofN-hydroxysuccinimide and 934 mg (4.55 mmol) of DCC were added thereto,followed by 2 hours of reaction at 40° C. The reaction liquid wasfiltered and then hexane was added until crystals were precipitated. Thecrystals were collected by filtration and again dissolved in 100 ml ofethyl acetate and 10 ml of acetonitrile. Then, hexane was added to thesolution until crystals were precipitated. The crystals were collectedby filtration and dried to obtain the following succinimide compound(p32).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 2.07 (2H, m,—OCOCH₃CH₂CH₂COON—), 2.50 (2H, t, —OCOCH₂CH₂CH₂COON—), 2.72 (2H, t,—OCOCH₂CH₂CH₂COON—), 2.84 (4H, s, succinimide), 3.38 (6H, s, —CH₃),3.40-3.80 (1771H, m, —CH₂—O(CH₂CH₂O)_(m)CH₃, CHO(CH₂CH₂O)_(m)CH₃),4.10-4.30 (2H, m, —CH₂OCOCH₂CH₂CH₂COON—).

Example 20

To a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 27.3 g (0.15 mol) of3-benzyloxy-1,2-propanediol, 135 g of dry toluene, and 0.9 g (39 mmol:26 mol %) of sodium. With introduction of nitrogen thereinto, the wholewas stirred at 80° C. until sodium dissolved. After dissolution, thesolution was further stirred at 80° C. for 2 hours.

The reaction liquid was charged into a 5 L autoclave thoroughly driedbeforehand and the same operations as in Examples 16-3, 16-6, 16-7, and16-8 were conducted to obtain the compound (p33) having the samestructure as that of (p28).

Example 21

The solution of 3-benzyloxy-1,2-propanediol alcoholated with sodium inExample 1-3, the solution of 3-benzyloxy-1,2-propanediol alcoholatedwith sodium in Example 16-3, and the solution of3-benzyloxy-1,2-propanediol alcoholated with sodium before charging intothe autoclave in Example 20 were sampled and converted into derivativesunder the following conditions, and then they were measured by gaschromatography (GC). The results are shown in Table 3.

Each sample was weighed in an amount of 0.2 g and dissolved with adding1.0 ml of pyridine, and then 0.8 ml of hexamethyldisilazane was addedthereto. To the solution was added 0.4 ml of chlorotrimethylsilane,followed by 30 minutes of stirring. The reaction liquid was filtratedthrough a syringe filter (PTFE, 0.45 μm) and GC measurement wasconducted under the following conditions:

GC system: HP6890, column: HP-5 (0.25 μm×30 cm), detector: FID,injection temperature: 320° C., injection: splitless, injection amount:0.2 μl, carrier gas: helium, flow rate: 23 cm/sec, column temperature80° C. (0 min)→15° C./min→320° C. 24 min), detector temperature: 320° C.

TABLE 3 Benzyl alcohol Glycerin Example 1-3 0% 0.4% Example 16-3 0% 0.3%Example 20 2.2%   1.3%

From the results of Table 3, it was found that benzyl which causesformation of reactive low-molecular-weight impurities and glycerin whichcauses formation of non-reactive high-molecular-weight impurities werehardly produced under the treating conditions with sodium as in Examples1-3 and 16-3.

Example 22

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 10 g (1 mmol) of the compound (p2) obtained in Example1-4 and 50 g of toluene, and the whole was heated under reflux to effectazeotropic removal of water. After cooling to room temperature, 2.02 g(20 mmol) of triethylamine was added thereto and the whole was heated to40° C. Then, 0.687 g (6 mmol) of methanesulfonyl chloride were addedthereto, followed by 3 hours of reaction at 40° C. After completion ofthe reaction, hydrochloride salt was removed by filtration and 100 ml ofethyl acetate was added to the filtrate, followed by addition of hexaneuntil crystals were precipitated. The resulting crystals were collectedby filtration and the crystals were dissolved in 200 ml of ethyl acetateunder heating. After cooling to room temperature, hexane was added untilcrystals were precipitated. The crystals were collected by filtrationand dried. Then, 20 mg of the resulting crystals was sampled anddissolved in deuterated chloroform, and ¹H-nuclear magnetic resonancemeasurement was conducted (integration: 128 times) to obtain a spectrum.At that time, Mme was 6 and Mms was 0.073.

Example 23

Using 10 g (0.5 mmol) of the compound (p12) obtained in Example 7-2,1.01 g (10 mmol) of triethylamine, and 0.344 g (3 mmol) ofmethanesulfonyl chloride, the same operations as in Example 22 wereconducted. Then, ¹H-nuclear magnetic resonance measurement was conducted(integration: 256 times) to obtain a spectrum. At that time, Mme was 6and Ms was 0.102.

Example 24

Using 1.0 g (0.5 mmol) of the compound (p24) which had been twicealkyl-etherified in Example 1.6-4, 1.01 g (10 mmol) of triethylamine,and 0.344 g (3 mmol) of methanesulfonyl chloride, the same operations asin Example 22 were conducted. Then, ¹H-nuclear magnetic resonancemeasurement was conducted (integration: 256 times) to obtain a spectrum.At that time, Mme was 6 and Mms was 0.019.

Example 25

Using 11.3 g (0.25 mmol) of the compound (p27) which had been twicealkyl-etherified in Example 16-7, 0.506 g (5 mmol) of triethylamine, and0.172 g (1.5 mmol) of methanesulfonyl chloride, the same operations asin Example 22 were conducted. Then, ¹H-nuclear magnetic resonancemeasurement was conducted (integrations 256 times) to obtain a spectrum.At that time, Mme was 6 and Mms was 0.026.

Example 26

Using Mme, Mms, and peak top molecular weight (Mp) obtained in each ofExamples 22 to 25, Hrd and Hrd/Mp×1000000 were calculated. As the peaktop molecular weight, each data of (p3), (p13), (p25), and (p28) wasused. The results are shown in Table 4. As a result, it was revealedthat the ratio of alkyl-etherification of the compound represented bythe formula (p) of the invention was high and, in the case that thealkyl-etherification was repeated, the conversion was even higher andthe hydroxyl group remained only a little.

TABLE 4 Hrd Mp Hrd/Mp × 1000000 Example 22 0.0120 10351 1.16 Example 230.0167 18989 0.88 Example 24 0.0032 19687 0.16 Example 25 0.0043 450570.10

Example 27

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 10 g (1 mmol) of the compound (p3) obtained in Example1-5 and 50 g of toluene, and the whole was heated under reflux to effectazeotropic removal of water. After cooling to room temperature, 2.02 g(20 mmol) of triethylamine was added thereto and the whole was heated to40° C. Then, 0.687 g (6 mmol) of methanesulfonyl chloride were addedthereto, followed by 3 hours of reaction at 40° C. After completion ofthe reaction, hydrochloride salt was removed by filtration and 100 ml ofethyl acetate was added to the filtrate, followed by addition of hexaneuntil crystals were precipitated. The resulting crystals were collectedby filtration and the crystals were dissolved in 200 ml of ethyl acetateunder heating. After cooling to room temperature, hexane was added untilcrystals were precipitated. The crystals were collected by filtrationand dried. Then, 20 mg of the resulting crystals was sampled anddissolved in deuterated methanol, and ¹H-nuclear magnetic resonancemeasurement was conducted (integration: 128 times) to obtain a spectrum.At that time, when M1 detected at 3.132 ppm was regarded as 3, M2detected at 3.117 ppm was 0.295.

Example 28

Using 10 g (0.5 mmol) of the compound (p25) obtained in Example 16-5,1.01 g (10 mmol) of triethylamine, and 0.344 g (3 mmol) ofmethanesulfonyl chloride, the same operations as in Example 27 wereconducted. Then, ¹H-nuclear magnetic resonance measurement was conducted(integration: 256 times) to obtain a spectrum. At that time, M1 was 3and M2 was 0.091.

Example 29

Using 11.3 g (0.25 mmol) of the compound (p28) obtained in Example 16-8,0.51 g (5 mmol) of triethylamine, and 0.172 g (1.5 mmol) ofmethanesulfonyl chloride, the same operations as in Example 27 wereconducted. Then, ¹H-nuclear magnetic resonance measurement was conducted(integration: 256 times) to obtain a spectrum. At that time, M1 was 3and M2 was 0.112.

Example 30

Using 11.3 g (0.25 mmol) of the compound (p33) obtained in Example 20,0.51 g (5 mmol) of triethylamine, and 0.172 g (1.5 mmol) ofmethanesulfonyl chloride, the same operations as in Example 27 wereconducted. Then, ¹H-nuclear magnetic resonance measurement was conducted(integration: 256 times) to obtain a spectrum. At that time, M1 was 3and M2 was 0.212.

Example 31

From M1 and M2 obtained in each of Examples 27 to 30, M2/(M1+M2)×100 wascalculated. The results are shown in Table 5. As a result, it wasrevealed that the compounds of the invention each had a high purity.Also, from the results of Examples 29 and 30, it was found that higherpurity was achieved by carrying out the alcoholation of the compoundrepresented by the formula (9) with the temperature being lowered.

TABLE 5 M1 M2 M2/(M1 + M2) × 100 Example 27 3 0.295 8.95 Example 28 30.091 2.94 Example 29 3 0.112 3.60 Example 30 3 0.212 6.60 Example 32 30.162 5.12

Example 32

Into a 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added100 g of the above compound (p27) and 200 g of 5% palladium-carbon (50%hydrous product, manufactured by N. E. N. Cat.) as the hydrous product,and debenzylation was carried out in the same manner as in Example 16-8to obtain the compound (p34) having the same structure as that of (p28).At that time, the water content in the reaction system was measured bymeans of Karl Fisher's moisture meter and found to be 4.17%.

Using 11.3 g (0.25 mmol) of the resulting compound (p34), 0.51 g (5mmol) of triethylamine, and 0.172 g (1.5 mmol) of methanesulfonylchloride, the same operations as in Example 27 were conducted. Then,¹H-nuclear magnetic resonance measurement was conducted (integration:256 times) to obtain a spectrum. At that time, M1 was 3 and M2 was0.162.

As shown in Table 5, from the results of Examples 29 and 32, it wasfound that more highly pure compound of the formula (p) could beobtained by reducing the water content in the reaction system to 1% orlower.

Example 33 Modification of Peptide

A peptide of Humanin(Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala)(molecular weight 2687.2) was adjusted to 0.5 μM with 10 mM phosphatebuffer (pH=6.4). Into 200 μl of the solution was added 4 mg of thecompound of the formula (p31), followed by 4 hours of reaction at roomtemperature. Then, 200 μl of the reaction liquid was charged into aSP-Sepharose FT (manufactured by Amersham) column, which was thenequilibrated with 20 mM Tris-HCl buffer (pH=8.2). After theequilibration, a solution obtained by adding NaCl to the buffer so as tobe 1N was passed through the column and a fraction of the peptidemodified with (p31) was obtained with monitoring the elute by UV.Thereafter, 20 μl of the fraction was mixed with 20 μl of a Tris-SDSsample-treating liquid, followed by heating on a boiling water bath for2 minutes and 30 seconds. Then, 20 μl of the solution was analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-20%). Thestaining was carried out by CBB staining. The results were shown in FIG.6.

As a result, it was found that the mercapto group (cysteine) of thepeptide reacted with the maleimide of (p31), thereby modification wasachieved.

Example 34 Synthesis of Succinimide Compound (Group I(a)) (Case ofR=Methyl Group, A²O, A²O=Oxyethylene Group, n=0, and MolecularWeight=about 45000)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 11.3 g (0.25 mmol) of the compound (p28), 0.1 g ofsodium acetate, and 100 ml of toluene, and the whole was refluxed toeffect removal of water. Then, 285 mg (2.5 mmol) of glutaric anhydridewas added to the reaction liquid, followed by 12 hours of reaction at110° C. After cooling of the reaction liquid, 518 mg (4.5 mmol) ofN-hydroxysuccinimide and 934 mg (4.55 mmol) of DCC were added thereto,followed by 2 hours of reaction at 40° C. The reaction liquid wasfiltered and then hexane was added until crystals were precipitated. Thecrystals were collected by filtration and again dissolved in 200 ml ofethyl acetate and 20 ml of acetonitrile. Then, hexane was added to thesolution until crystals were precipitated. The crystals were collectedby filtration and dried to obtain the following succinimide compound(p35).

¹H-NMR (CDCl₃, internal standard: TMS) δ (ppm): 2.07 (28, m, —OCOCH₂ CH₂CH₂COON—), 2.50 (2H, t, —OCOCH₂ CH₂CH₂COON—), 2.72 (2H, t, —OCOCH₂CH₂CH₂ COON—) 2.84 (4H, s, succinimide), 3.38 (6H, s, —CH₃ ), 3.40-3.80(4043H, m, —CH₂ O(CH₂CH₂ O)_(m)CH₃, CHO(CH₂CH₂ O)_(m)CH₃) 4.10-4.30 (2H,m, —CH₂ OCOCH₂CH₂CH₂CH₂COON—).

Example 35 Modification of Insulin

Using the succinimide of (p32) obtained in Example 19 and thesuccinimide of (p35) obtained in Example 34, insulin (recombinant humaninsulin, Mw 5800, manufactured by SEROLOGICALS CORPORATION) wasmodified.

Using 0.1N sodium carbonate buffer (pH=9.0), a 10 mg/ml buffer solutionof the insulin was prepared. Into 100 μl of the solution was added 6.8mg of the compound of the formula (p32), followed by 20 hours ofreaction at 4° C. Then, the whole amount of the reaction liquid wascharged into a Q-Sepharose FF (manufactured by Amersham) column, whichwas then equilibrated with 20 mM Tris-HCl buffer (pH=8.2). After theequilibration, a solution obtained by adding NaCl to the buffer so as tobe 1N was passed through the column and a fraction of the peptidemodified with (p32) was obtained with monitoring the elute by UV.Thereafter, 20 μl of the fraction was mixed with 20 μl of a Tris-SDSsample-treating liquid, followed by heating on a boiling water bath for2 minutes and 30 seconds. Then, 20 μl of the solution was analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-20%). Thestaining was carried out by CHB staining.

Similarly, in the case of (p35), 13.6 mg of the compound of the formula(p35) was added to 100 μl of a 10 mg/ml buffer solution of the insulinand the whole was treated in a similar manner.

The results were shown in FIG. 7. As a result, it was found that theinsulin was modified with the compound of the formula (p32) or (p35).

1. A process for producing a polyalkylene glycol derivative of theformula (11), comprising the following step (AA): Step (AA): a step ofsubjecting a compound represented by the formula (10) to a hydrogenativereduction reaction under a condition that a water content in a reactionsystem is 1% or less:

wherein G is a residual group of a compound having 2 to 4 hydroxylgroups; R² is a hydrocarbon group having 1 to 4 carbon atoms; m1, m2,and m3 represent each average number of moles of an oxyethylene groupadded and satisfy the following relationship:0≦m1≦1000, 0≦m2≦1000, 0≦m3≦1000, 10≦m1+m2+m3≦1000; X¹ is an amino group,a carboxyl group, or a protected group thereof; and g1, g2, and g3represent each an integer and satisfy the following relationalequations:1≦g1≦3, 0≦g2, 0≦g3, 2≦g1+g2+g3≦4.
 2. A process for producing apolyalkylene glycol derivative of the formula (11) according to claim 1,wherein in the step (AA), palladium is used as a hydrogenative reductioncatalyst, palladium is added in an amount of 1 to 20 wt % based on thecompound of the formula (10), and the reaction is carried out at atemperature of 40° C. or lower.
 3. A process for producing apolyalkylene glycol derivative of, the formula (16), wherein thefollowing steps (BB1) and (BB2) are carried out: Step (BB1): a step ofadding a dehalogenating agent and a compound represented by the formula(14) to a compound represented by the formula (12) and reacting them at20 to 60° C. to obtain a compound of the formula (13), provided thateach charged molar ratio satisfies the following relationship:Vj≧1.5×Vh×g5Vi>Vj Vh: number of moles of the compound represented by the formula(12) Vi: number of moles of the dehalogenating agent Vj: number of molesof the compound represented by the formula (14); Step (BB2): a step ofadding a compound represented by the formula (15) to the compound of theformula (13) and reacting them at 20 to 80° C. to obtain a compound ofthe formula (16), provided that each charged molar ratio satisfies thefollowing relationship:Vk>Vj Vk: number of moles of the compound represented by the formula(15):

wherein G is a residual group of a compound having 2 to 4 hydroxylgroups; R² is a hydrocarbon group having 1 to 4 carbon atoms; m1, m2,and m3 represent each average number of moles of an oxyethylene groupadded and satisfy the following relationship:0≦m1≦1000, 0≦m2≦1000, 0≦m3≦1000, 10m1+m2+m3≦1000; X¹ is an amino group,a carboxyl group, or a protected group thereof; g4, g5, and g6 representeach an integer and satisfy the following relational equations:0≦g4, 1≦g5≦3, 0≦g6, 2≦g4+g5+g6≦4; W is a halogen atom selected from Cl,Br and I; R³ is a hydrocarbon group having 1 to 10 carbon atoms; and Mis potassium or sodium.
 4. The process for producing a polyalkyleneglycol derivative of the formula (16) according to claim 3, comprising astep (BB3) as a successive step of the step (BB2): Step (BB3): a step offiltrating the reaction liquid or washing the reaction liquid with anaqueous inorganic salt solution having a concentration of 10 wt % ormore.
 5. The process for producing a polyalkylene glycol derivative ofthe formula (16) according to claim 4, wherein the steps (BB1) to (BB3)are repeated after the step (B3).